Peptides and methods of using same

ABSTRACT

We describe peptides and their uses for the treatment of autoimmune, inflammatory and metabolic diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application under 35 U.S.C. § 121 of aco-pending U.S. application Ser. No. 13/925,067, which is acontinuation-in-part application of an International Application No.PCT/US13/20498 filed Jan. 7, 2013, which claims benefit under 35 U.S.C.§ 119(e) of provisional application No. 61/584,517 filed Jan. 9, 2012and provisional application No. 61/699, 571 filed Sep. 11, 2012, thecontents of which are incorporated herein by reference in theirentirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing, which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 31, 2012, isnamed 65292741.txt and is 18,884 bytes in size.

FIELD OF THE INVENTION

The present disclosure presents isolated and/or synthesized peptides.Specifically, the present invention is a class of isolated and/orsynthesized peptide fragments, and synthetic analogs based on thepeptide fragments. The peptides can have preventative and therapeuticeffects in human disease and can be used in treatment of human disease.

BACKGROUND

Serine protease inhibitors (Serpins) represent a large (>1000) family ofprotease inhibitors, present in all branches of life and involved in amultitude of physiological processes. In mammals, such as humans,Serpins are important for homeostasis and although a certain level ofpromiscuity exists, each Serpin has a cognate serine protease(s). Forexample, alpha-1-antitrypsin (AAT) and alpha-1-antichymotrypsin (ACT)inhibit inflammatory proteases such as elastase, whereas antithrombininhibits thrombin and plays a role in coagulation.

A number of specific AAT mutations are manifested in human disease,including COPD, thrombosis and Serpinopathies (cirrhosis and dementia).Currently, a small number of human serum-derived AAT formulations areapproved by the FDA for treatment of COPD. In this therapeutic approach,AAT functions as a protease inhibitor similar to endogenous AAT.

AAT is the archetypical Serpin and shares tertiary structure with otherSerpins. Serpins have a ˜20 amino acid (aa) exposed loop, called thereactive center loop (RCL), which serves as bait for the cognateproteases. Once the protease binds the RCL, it becomes trapped,partially unfolded and destined for degradation. The cleavage of the RCLat its P1-P1′ site drives the process of protease inactivation andresults in the release of a small C-terminal peptide from the Serpinmolecule.

SUMMARY

We have unexpectedly found that many of the C-terminal peptides from theSerpin molecule and variants and derivatives thereof function as potentanti-inflammatory agents. Accordingly, we provide peptide compositions,pharmaceutical compositions comprising the C-terminal Serpin peptidesand methods of using the peptides to treat inflammatory and autoimmuneconditions including type II diabetes, lupus and graft versus hostdisease, uveitis, eczema and psoriasis, cystic fibrosis, rheumatoidarthritis (RA), acute radiation syndrome and burn patients, inflammatorybowel disease (IBD) and new onset type I diabetes.

The invention is based on our finding that a short peptide, SP16 (SEQ IDNO: 1) derived from human alpha-1-antitrypsin shows anti-inflammatoryand immune-modulatory properties similar to the much larger parentprotein, alpha-1-antitrypsin. Without wishing to be bound by a theory,SP16 appears to be a first-in-class peptide master switch for treatmentof autoimmune, inflammatory and metabolic diseases.

Specifically, we have shown the function of the SP16 peptide consistingof an amino acid sequence VKFNKPFVFLMIEQNTK (SEQ ID NO: 1) inwell-established animal models for at least type II diabetes, rheumatoidarthritis, and lethal endotoxemia.

Accordingly, we provide a composition comprising an isolated peptidecomprising, consisting essentially of, or consisting of the amino acidsequence X1-Z1-F-N-K-P-F-X2-Z2-X3-Z3-Q (SEQ ID NO: 2), wherein

X1 is V or L;

X2 is V, L or M;

X3 is M, I or V;

Z1 is any amino acid;

Z2 is a sequence of any two amino acids; and

Z3 is a sequence any five amino acids, and wherein the isolated peptideconsists of 37 or fewer amino acids.

The peptide can be modified to extend the shelf life and/orbioavailability using one or more non-natural peptide bonds or aminoacids or by attaching to the peptide functional groups such as, e.g.,polyethylene glycol (PEG).

The composition may further comprise a carrier, such as apharmaceutically acceptable carrier.

The peptides of the invention can be used to reduce the serum TNF-αlevels in human individuals who have pathologically increased TNF-αlevels. Thus the invention provides a method or use for reducing TNF-αlevels in a human in need thereof comprising administering to the humanindividual the peptide of the invention in a pharmaceutically acceptablecarrier. In certain embodiments, the isolated peptide results in a 50%or 75% decrease in serum TNF-α levels when administered in an effectiveamount to a human subject compared to the levels before administrationof the isolated polypeptide. In other embodiments, the isolated peptidefurther comprises at least one other protein. The combination of the atleast two proteins can be referred to as a fusion protein. The otherprotein can be selected from an epitope tag and a half-life extender.The peptide can comprise both an epitope tag and a half-life extender.

We also provides a composition comprising an isolated peptide consistingessentially of or consisting of the amino acid sequenceX1-Z1-F-N-X2-P-F-X3-Z2-X4-Z3-X5 (SEQ ID NO: 3), wherein

X1 is V or L;

X2 is K or R;

X3 is V, L or M;

X4 is M, I or V;

X5 is K or Q;

Z1 is any amino acid;

Z2 is a sequence of any two amino acids;

Z3 is a sequence any five amino acids; and

wherein the isolated peptide causes a 75% decrease in serum TNF-α levelswhen administered in an effective amount to a human subject compared tothe amount of TNF-α levels before administering the peptide.

In certain embodiments, the isolated peptide comprises the amino acidsequence X1-Z1-F-N-X2-P-F-X3-Z2-X4-Z3-X5 (SEQ ID NO: 3), wherein X1, X2,X3, X4, X5, Z1, Z2, and Z3 are defined as above and wherein the peptideconsists of, at most, 35, 22 or 21 amino acid residues.

In certain aspects, the peptides of any of the embodiments describedherein and throughout the specification, also comprise at least oneother protein. The combination of these at least two proteins can bereferred to as a fusion protein. Specifically the other protein can beselected from an epitope tag and a half-life extender. In some aspectsof all the embodiments of the invention, the isolated peptide cancomprise both an epitope tag and a half-life extender.

The disclosure also provides an isolated peptide consisting essentiallyof or consisting of the amino acid sequence RFNRPFLR (SEQ ID NO: 4) andRFNKPFLR (SEQ ID NO: 5). In certain embodiments, the isolated peptidecauses a 50% or 75% decrease in serum TNF-α levels compared to theamount of TNF-α levels before administering the peptide whenadministered in an effective amount to a human subject.

In other aspects of all the embodiments of the invention, the isolatedpeptide is linked another protein. The combination of these proteins canbe referred to as a fusion protein. Specifically the other protein canbe selected an epitope tag and a half-life extender.

In some aspects of all the embodiments of the invention, the isolatedpeptide consists of, at most, 100, 35, 22, 21 or 9 additional aminoacids.

In other embodiments, the isolated peptide consists essentially of, orconsists of the amino acid sequence of Z1-RFNRPFLR-Z2 (SEQ ID NO: 6) andZ1-RFNKPFLR-Z2 (SEQ ID NO: 7), wherein Z1 and Z2 are independently 1, 2,3, 4, 5, 6, 6, 7, 8, 9, 10 or between 1 and 3, between 1 and 5, between1 and 6, between 1 and 7, between 1 and 8, between 1 and 9, or between 1and 10 basic amino acids.

In some embodiments, the isolated peptide consists essentially of orconsists of the amino acid sequence of RRRFNRPFLRRR (SEQ ID NO: 8) andRRRFNKPFLRRR (SEQ ID NO: 9).

The disclosure also provides a composition comprising an isolatedpeptide consisting essentially of or consisting of the amino acidsequence of FNRPFL (SEQ ID NO: 10) and FNKPFL (SEQ ID NO: 11).

The disclosure also provide a composition comprising an isolated orsynthesized peptide consisting essentially of or consisting of any oneor a combination of the following peptides: SP40; SP43; SP46; and SP49as set forth in Table B, and their use in methods for treatinginflammation, rheumatoid arthritis, COPD, cystic fibrosis, improvingglycemic control in diabetic subjects and preventing and treatingendotoxemia, for example, in burn victims and subjects exposed to acuteradiation.

In one embodiment, the disclosure also provides a method of decreasingserum TNF-α compared to the amount of TNF-α levels before administeringthe peptide to a subject comprising administering to the subject aneffective amount of any one of the isolated peptides as defined above todecrease the serum TNF-α levels by at least 50%. In one embodiment,serum TNF-α levels are decreased by 75% compared to the amount of TNF-αlevels before administering the peptide. In other embodiments, thesubject is a mammal. In some aspects of all the embodiments of theinvention, the mammal is a human.

In some embodiments, the disclosure provides methods of improvingglycemic control or reducing hyperglycemia in a subject in need thereofcomprising administering to the subject with hyperglycemia any of thepeptides described herein in a pharmaceutically acceptable carrier.

In some aspects of all the embodiments of the invention, the human hasbeen diagnosed with type II diabetes, new onset type I diabetes,rheumatoid arthritis, COPD, cystic fibrosis, uveitis, eczema, psoriasis,lupus, graft versus host disease, inflammatory bowel disease (IBD), orendotoxemia following acute radiation exposure or burn prior toadministering the peptide.

In some aspects, the human has not been subjected to prior treatmentwith alpha antitrypsin, such as alpha-1-antitrypsin treatment before thetreatment with the peptides of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a table showing the sequences and homology of SerpinC-terminal peptides (SEQ ID NOs: 18-24, respectively, in order ofappearance).

FIG. 2 is a table showing peptides derived from truncations (SEQ ID NOs:25-32, 1, 33-34, 1, 33, 38-51, 10, and 8, respectively, in order ofappearance).

FIG. 3 is a bar graph showing TNF-α levels in blood of mice injectedwith 0.5, 0.1, 0.02 or 0.004 mg of various peptides (SP1-SP18 from leftto right on the x-axis). Each peptide was administered in four differentconcentrations. The four bars for each of the peptide from left to rightrepresent the concentrations from highest (0.5 mg) to the lowest (0.004mg).

FIG. 4 is a bar graph showing TNF-α levels in blood of mice injectedwith 0.004 mg of various peptides.

FIG. 5 is a line graph showing cumulative paw scores for mock treated,dexamethasone treated and peptide (SEQ ID NO: 1, also referred to asSP16) in a collagen antibody induced arthritis (CAIA) rat model.

FIG. 6 is a line graph showing cumulative paw scores for mock treated,dexamethasone treated and peptide (SEQ ID NO: 1, also referred to asSP16) in a collagen antibody induced arthritis (CAIA) rat model.

FIG. 7 is a graph summarizing survival during a lethal endotoxemia studyin mice. Animals were injected with 0.6 mg/kg SP16 (SEQ ID NO: 1) asindicated: 2 hours before, at the time of, and/or 0.5 hours afterinduction of Sepsis. Sepsis was induced by injection of 15 mg/kg LPS atT=0. Survival was monitored at the indicated time points. Survival at 72hours was 60% in the group that received SP16 at T=−2, 0 and 0.5 hours.

FIGS. 8A-8F show graphs summarizing data from a study in the db/db T2DMmodel. Five-week old db/db mice were assigned to groups of 10 animals,and IP injected with 0.6 mg/kg SP16 (biweekly), 15 mg/kg Rosiglitazone(biweekly), or vehicle control for 5 weeks. HbA1c (FIG. 8A) andC-peptide (FIG. 8B) levels were determined at the end of the study.During the last week of the study, a glucose tolerance test wasadministered (FIG. 8C). Non-fasted blood glucose was measured twice aweek throughout the study and was significantly lowered in the SP16 (SEQID NO: 1) and Rosiglitazone groups. Values represent averages, withstandard errors indicated. (*) indicates P<0.05 and (**) p<0.01,compared to the Mock group by a T-test. FIG. 8D summarizes the extent ofislet hyperplasia in the db/db study as assessed by morphometry. FIGS.8E and 8F summarize serum CRP and TGF-beta levels in the db/db mousemodel of type II diabetes. Decreased serum CRP levels are consistentwith peptide treatment promoting an anti-inflammatory cytokine profile.Eight week old diabetic db/db mice were assigned to groups of 8. Groupreceived Saline (Mock) or 0.6 mg/kg SP16 biweekly for 12 weeks. Pooledserum CRP and TGF-beta levels were determined for each group. (*)indicates p<0.05 and (**) p<0.01 compared to the Vehicle control group.

FIG. 9 is a graph summarizing data from a study in the mouse CAIARheumatoid Arthritis model. The graph shows cumulative swelling scoresfor all paws at the peak of disease (Day 7) for groups of 5 animals.Balb/c mice were IV injected with a collagen antibody cocktail (MDBioSciences) on Day 0 and IP injected with LPS on Day 3. Normal ControlAnimals received no injections and served as disease-free baselinecontrol. Daily SP16 injection provided protection equivalent toDexamethasone.

FIGS. 10A and B. FIG. 10A presents exemplary peptides that were used toreduce TNF-alpha levels in mice subjected to an LPS challenge. FIG. 10Bshows TNF-alpha levels in LPS challenged mice. The mice were treatedwith alanine scanned SP16 peptide. Dexamethasone served as positivecontrol of “effective treatment” and the peptides SP16, SP40, SP43, SP46and SP49 all reduce TNF-alpha levels more than Dexamethasone. Based onthis alanine screen, and without wishing to be bound by a theory, itappears that the C- and N-terminal amino acids contribute to theanti-inflammatory effect of the SP16 peptide. Compare to FIG. 4 whichshows a graph summarizing serum TNF-alpha levels in a mouse inflammationmodel, LPS challenge, treated with different human Serpin derivedpeptides. Groups of 3 animals were injected with 0.2 mg/kg peptide andsubjected to LPS challenge. Serum TNF-alpha levels were determined byELISA. Several peptides provided the same level of protection as 1 mg/kgDexamethasone.

FIG. 11 shows that SP16 is a TLR2 agonist. Graph summarizingrepresentative data from assays with an engineered TLR-2 indicator cellline (HEK-Blue™ mTLR2, Invivogen). Cells were incubated with theindicated concentrations of peptide for 24 hours. Upon TLR2 activation,the cells secrete alkaline phosphatase which can be assayed. The assaywas done in triplicate and averages with standard deviations areplotted. SP16 exhibited TLR-2 ligand properties, inducing TLR-2signaling in a dose dependent manner. The SP34 scrambled control peptideshowed no TLR2 induction. *p<0.05, compared to scrambled control (SP34).

FIG. 12 shows structure activity relationship analysis for SP16. Graphsummarizing data from an experiment where an engineered TLR-2 indicatorcell line (HEK-Blue™ mTLR2, Invivogen) was used to test the impact ofsubstituting amino acid residues of the SP16 peptide with alanine(“alanine scan”). Cells were incubated with 20 μg/ml of the indicatedpeptides for 24 hours. Upon TLR2 activation, the cells secrete alkalinephosphatase which can be assayed. The assay was done in triplicate andaverages are plotted. Peptide sequences are shown in the followingfigure. *p<0.05, compared with scrambled control (SP34).

FIG. 13 shows Structure activity relationship analysis for SP16. Tableshowing the amino acid sequences (SEQ ID NOS 1, 18, 12-17, 52-56, and51, respectively, in order of appearance) of peptides that were testedusing a TLR-2 indicator cell line (See data in previous figure). Theright side of the table summarizes the peptides' impact on TLR-2signaling (* indicates low, ***** indicates high, N/A had no impact onsignaling) The data suggest the first three residues contribute toinducing TLR-2 signaling. If residues 1-3 are substituted with alanines(SP37), the mutant peptide has no impact on TLR2. However, whensubstituted individually (SP52-SP54), the peptides retain the ability tostimulate TLR-2. Surprisingly, substitution of the phenyl alanineresidue at position 3 with a smaller alanine residue enhances theability to stimulate TLR-2 signaling compared to SP16.

FIG. 14 shows a schematic of the different phases of Type II diabetes.

FIG. 15 shows a graph summarizing data from an experiment with primaryhuman synoviocytes. Cells were incubated with 10 uM of the indicatedpeptides, as well as 0, 5, 10, or 30 ng/ml IL1b, for 48 hours. Upon IL1bstimulation, the cells secrete the metaloprotease MMP1, which isinvolved in breaking down the cartilage in Arthritis. The assay was donein triplicate and averages with standard deviations are plotted. SP16lowered MMP1 secretion compared to the scrambled control peptides, at 20ng/ml IL1b. (*) indicates p<0.05 compared to scrambled peptide control.

FIG. 16 shows a graph summarizing data from a study in the mouse CAIARheumatoid Arthritis model. The graph shows the average cumulativeswelling (clinical) scores for all paws at the peak of disease (Day 7)for groups of 5 animals. Balb/c mice were IV injected with a collagenantibody cocktail (MD BioSciences) on Day 0 and IP injected with LPS onDay 3. Normal Control Animals received no injections and served asdisease-free baseline control. SP16 was provided daily byintraperitoneal injection (Dose: 12 ug/animal) or by oral gavage (Dose:25 and 50 ug/animal). Daily SP16 injection provided protectionequivalent to daily administration of 1 mg/kg Dexamethasone.

FIG. 17 depicts a graph with data from an experiment with mouse RAW(macrophage) cells. Cells were incubated with 20 or 40 ug/ml of SP16peptide, as well as 0, 2.5, 5 or 10 ng/ml LPS, for 24 hours. Upon LPSstimulation, the cells secrete the inflammatory cytokine IL-6, which wasmeasured by ELISA. The assay was done in triplicate and averages withstandard deviations are plotted. SP16 lowered LPS-induced IL-6 secretionconsistent with its anti-inflammatory function. (*) Indicates p<0.05compared to scrambled peptide control.

FIG. 18 shows data from a study in NOD mice, a spontaneous autoimmunediabetes model. Groups of 12 animals were injected with SP16, hAAT orscrambled control peptide. Non-fasted blood glucose levels were measuredtwice a week and animals with two consecutive readings greater than 300mg/dl were considered diabetic. Both SP16 and hAAT delayed and reducedthe incidence of diabetes.

DETAILED DESCRIPTION

The present disclosure describes isolated peptides used and useful inthe field of disease treatment and prevention. Specifically, the presentinvention provides isolated and/or synthesized peptides, and syntheticanalogs based on these peptides, with preventative and therapeuticeffects in human disease. For example, the peptides may be used to treator prevent inflammation, auto-immune tissue destruction, e.g., in lupusand host versus graft disease, rheumatoid arthritis, cystic fibrosis,eczema, psoriasis and to treat type II and type I diabetes, for exampleto stimulate expansion of beta cell mass in an individual with diabetes,to treat inflammatory bowel disease as well as to treat or preventendotoxemia following acute radiation exposure and in burn patients.

The peptides described herein are specifically defined short isolated orsynthesized C-terminal peptides based on Serpins and variants andderivatives thereof with surprisingly effective anti-inflammatoryproperties and with much more useful size for therapeutic applicationscompared to the native Serpin proteins. The isolated peptides are shownin FIGS. 1-2. FIG. 1 shows the amino acid sequences of the C-terminalfragments of a variety of Serpins. Each peptide is marked with a SEQ IDNO: in column 2, immediately to the left of the peptide. FIG. 2 showstruncations of the C-terminal fragments shown in FIG. 1, as well asvariants and derivatives thereof. Again, each peptide is marked with aSEQ ID NO: in column 2, immediately next to the peptide.

We have discovered that SP16 (SEQ ID NO: 1), which is derived from humanalpha-antitrypsin exhibits anti-inflammatory and immune-modulatoryproperties similar to those of the parent protein, alpha-1-antitrypsin.SP16 appears to be a fist-in-class peptide master switch for treatmentof autoimmune, inflammatory and metabolic diseases. Without wishing tobe bound by a theory, the peptides of the invention can provide a goodsafety profile, based on the good safety profile of the parent protein,alpha-1-antitrypsin. However, the peptides of the invention are fareasier and thus less expensive to produce as they are much smaller thanthe parent protein.

We have discovered that C-terminal peptides that result from a Serpinmolecule's cleavage by one of its cognate serine proteases haveintrinsic biologic function that is distinct from that of the proteaseinhibitor function of the parent, complete Serpin molecule. For example,the C-terminal peptides from AAT, antichymotrypsin and Kallistatin havevarying degrees of anti-inflammatory effects. Based on our research, wesubmit that these anti-inflammatory and/or immune modulating peptides,that are a byproduct from the lifecycle of a Serpin molecule, representa type of an immunological and inflammatory (homeostatic) “masterswitch.”

Moreover, our data from engineered cell lines show that SP16 activatesthe TLR-2 signaling pathway. This is interesting because another immunemodulating peptide, DiaPep277, which shares no sequence similarity withSP16, has a similar TLR activation profile. Without wishing to be boundby a theory, based on these observations, we suggest that SP16 actsthrough the TLR2 receptor, and possible the T-cell receptor, to drivecytokine secretion to a Th2 anti-inflammatory cytokine profile (IL-4 andIL-10). In autoimmune diseases, SP16 is predicted to induce expansion ofregulatory T-cell populations and thereby shift the inflammatoryresponse towards a regulatory response.

We therefore provide novel anti-inflammatory molecules from theC-terminal peptides of Serpin molecules, and novel ways of developingadditional anti-inflammatory molecules by modifying the C-terminalfragments of Serpins.

The previously known functions of Serpins are related to inhibiting thefunction of serine protease enzymes. A few Serpins inhibit other typesof proteins, and several do not have an inhibitory function.

Serpins are a large family (>1000) of Serine Protease Inhibitors thatare structurally similar but functionally diverse. They are involved ina multitude of physiological processes and are critical for homeostasisin mammals. Genetic mutations in individual Serpins are manifested indifferent human diseases, including COPD, thrombosis and emphysema.

Each serpin with an inhibitory role is responsible for blocking theactivity of one or more proteins. Serpins bind to their target proteinsto prevent them from completing any further reactions. Upon binding to atarget, an irreversible change in the structure of a serpin proteinoccurs. Certain cells recognize when a Serpin is bound to its target andclear these attached proteins from the bloodstream.

Alpha-1-antitrypsin (AAT) is the prototypical Serpin. PROLASTIN®(Talecris), ZEMAIRA® (Aventis Behring) and ARALAST® (Baxter) are humanserum-derived AAT formulations approved by the FDA for treatment ofCOPD. AAT is currently in clinical trials for treatment of new onsettype I diabetes, graft vs. host disease and cystic fibrosis.

Researchers have identified at least 37 different serpin genes inhumans. Based on our research, we submit that isolated and synthesizedC-terminal fragments of the serpins proteins provide a novel source ofanti-inflammatory molecules. Thus, we submit that the C-terminalfragments of at least the Serpins listed in Table A are useful asanti-inflammatory molecules.

TABLE A Approved Symbol Approved Name Synonyms SERPINA1 serpin peptidaseinhibitor, clade A (alpha-1 AAT, A1A, PI1, alpha-1-antitrypsin,antiproteinase, antitrypsin), member 1 A1AT, alpha1AT SERPINA2 serpinpeptidase inhibitor, clade A (alpha-1 ATR, ARGS antiproteinase,antitrypsin), member 2 SERPINA3 serpin peptidase inhibitor, clade A(alpha-1 ACT, alpha-1-antichymotrypsin antiproteinase, antitrypsin),member 3 SERPINA4 serpin peptidase inhibitor, clade A (alpha-1 KST, KAL,KLST, kallistatin antiproteinase, antitrypsin), member 4 SERPINA5 serpinpeptidase inhibitor, clade A (alpha-1 PAI3, PROCI antiproteinase,antitrypsin), member 5 SERPINA6 serpin peptidase inhibitor, clade A(alpha-1 antiproteinase, antitrypsin), member 6 SERPINA7 serpinpeptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin),member 7 AGT angiotensinogen (serpin peptidase inhibitor, clade A,member 8) SERPINA9 serpin peptidase inhibitor, clade A (alpha-1CENTERIN, SERPINA11b, GCET1 antiproteinase, antitrypsin), member 9SERPINA10 serpin peptidase inhibitor, clade A (alpha-1 PZI, ZPIantiproteinase, antitrypsin), member 10 SERPINA11 serpin peptidaseinhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 11SERPINA12 serpin peptidase inhibitor, clade A (alpha-1 OL-64, Vaspinantiproteinase, antitrypsin), member 12 SERPINA13 serpin peptidaseinhibitor, clade A (alpha-1 UNQ6121 antiproteinase, antitrypsin), member13 (pseudogene) SERPINB1 serpin peptidase inhibitor, clade B EI, PI2,anti-elastase (ovalbumin), member 1 SERPINB2 serpin peptidase inhibitor,clade B HsT1201 (ovalbumin), member 2 SERPINB3 serpin peptidaseinhibitor, clade B T4-A, HsT1196 (ovalbumin), member 3 SERPINB4 serpinpeptidase inhibitor, clade B PI11, LEUPIN, SCCA-2, SCCA1 (ovalbumin),member 4 SERPINB5 serpin peptidase inhibitor, clade B maspin(ovalbumin), member 5 SERPINB6 serpin peptidase inhibitor, clade B PTI,CAP (ovalbumin), member 6 SERPINB7 serpin peptidase inhibitor, clade BMEGSIN (ovalbumin), member 7 SERPINB8 serpin peptidase inhibitor, cladeB CAP2 (ovalbumin), member 8 SERPINB9 serpin peptidase inhibitor, cladeB CAP3 (ovalbumin), member 9 SERPINB10 serpin peptidase inhibitor, cladeB bomapin (ovalbumin), member 10 SERPINB11 serpin peptidase inhibitor,clade B EPIPIN (ovalbumin), member 11 (gene/pseudogene) SERPINB12 serpinpeptidase inhibitor, clade B YUKOPIN (ovalbumin), member 12 SERPINB13serpin peptidase inhibitor, clade B HUR7, hurpin, headpin (ovalbumin),member 13 SERPINC1 serpin peptidase inhibitor, clade C ATIII, MGC22579(antithrombin), member 1 SERPIND1 serpin peptidase inhibitor, clade D(heparin HC-II, HLS2, HC2, D22S673 cofactor), member 1 SERPINE1 serpinpeptidase inhibitor, clade E (nexin, PAI plasminogen activator inhibitortype 1), member 1 SERPINE2 serpin peptidase inhibitor, clade E (nexin,PN1, GDN, PNI, nexin plasminogen activator inhibitor type 1), member 2SERPINE3 serpin peptidase inhibitor, clade E (nexin, plasminogenactivator inhibitor type 1), member 3 SERPINF1 serpin peptidaseinhibitor, clade F (alpha-2 EPC-1, PIG35 antiplasmin, pigment epitheliumderived factor), member 1 SERPINF2 serpin peptidase inhibitor, clade F(alpha-2 API, ALPHA-2-PI, A2AP, AAP antiplasmin, pigment epitheliumderived factor), member 2 SERPING1 serpin peptidase inhibitor, clade G(C1 C1IN, C1-INH, HAE1, HAE2 inhibitor), member 1 SERPINH1 serpinpeptidase inhibitor, clade H (heat HSP47, colligen shock protein 47),member 1, (collagen binding protein 1) SERPINI1 serpin peptidaseinhibitor, clade I neuroserpin (neuroserpin), member 1 SERPINI2 serpinpeptidase inhibitor, clade I PANCPIN, TSA2004, MEPI, pancpin (pancpin),member 2

We further performed an alanine screen that showed that isolated orsynthesized or modified SP16 peptides shown in Table B below areparticularly effective in reducing TNF-alpha levels in a mouse model forinflammation. Specifically, we discovered that in these particularfragments, the three most N-terminal and the two most C-terminal aminoacids appear to play a role in the anti-inflammatory properties of thepeptides as replacement of them appeared to reduce the capacity of thepeptides to reduce TNF-alpha levels in a LPS challenged mouse model ofsepsis (FIG. 10). Accordingly, in some aspects of all embodiments of theinvention the peptides are selected from SP40, SP43, SP46, and SP49 thepeptide sequences of which are set forth in Table B.

Table B shows peptides named SP16; SP40; SP43; SP46; and SP49 providedparticularly good anti-inflammatory effect when administered to a mousemodel of sepsis (See FIG. 10).

TABLE B Peptide Amino Acid Sequence SP16V K F N K P F V F L M I E Q N T K (SEQ ID NO: 1) SP37A A A N K P F V F L M I E Q N T K (SEQ ID NO: 12) SP40V K F A A A F V F L M I E Q N T K (SEQ ID NO: 13) SP43V K F N K P A A A L M I E Q N T K (SEQ ID NO: 14) SP46V K F N K P F V F A A A E Q N T K (SEQ ID NO: 15) SP49V K F N K P F V F L M I A A A T K (SEQ ID NO: 16) SP51V K F N K P F V F L M I E Q A A A (SEQ ID NO: 17)

According to some embodiments and aspects of the invention, any ofisolated peptides consisting of or consisting essentially of sequencesset forth in SEQ ID NOs: 8, 10, 19-34, and 38-49 can be used to reduceinflammation. Any of peptides consisting of or consisting essentially ofsequences set forth SEQ ID NOs: 8, 10, 19-34, and 38-49 can also be usedto reduce TNF-α in a subject. In certain embodiments, the amount ofTNF-α in the serum is reduced by up to 50% or more or 75% or morecompared to the amount of the same in the serum prior to administeringthe peptide.

Table C, provided at FIG. 10A, presents additional exemplary peptidesthat were used to reduce TNF-alpha levels in mice subjected to an LPSchallenge. See also FIG. 10B. The peptides with capacity to reduceTNF-alpha levels as shown in FIG. 10B are contemplated for thecompositions, pharmaceutical compositions and methods of use andtreatment of inflammatory conditions in the present invention.

SP1 (SEQ ID NO: 18); SP2 (SEQ ID NO: 19); SP3 (SEQ ID NO: 20); SP4 (SEQID NO: 21); SP5 (SEQ ID NO: 22); SP6 (SEQ ID NO: 23); SP7 (SEQ ID NO:24); SP8 (SEQ ID NO: 25); SP9 (SEQ ID NO: 26); SP10 (SEQ ID NO: 27);SP11 (SEQ ID NO: 28); SP12 (SEQ ID NO: 29); SP13 (SEQ ID NO: 30); SP14(SEQ ID NO: 31); SP15 (SEQ ID NO: 32); SP16 (SEQ ID NO: 1); SP17 (SEQ IDNO: 33); SP18 (SEQ ID NO: 34).

The phrase “consisting essentially of” is herein meant to define thescope of the peptides to the specified material amino acids, and to onlyinclude additional amino acids or changes that do not materially affectthe claimed invention's basic and novel characteristics, namely, theanti-inflammatory capacity of the short isolated or synthesizedpeptides. The definition specifically excludes peptides that have asequence of a complete Serpin protein, and the definition alsospecifically excludes peptide sequences that are equal to or longer than37 amino acids of any naturally occurring Serpin protein.

Without wishing to be bound by a theory, we have also identified theimportant amino acids that provide the core, and the possiblemodifications, for anti-inflammatory peptides as manufactured herein.Therefore, the isolated peptides encompassed by the formulae set forthbelow are also provided and they can be used to reduce inflammation.

Human AAT, antichymotrypsin, and kallistatin have been known to containelements with anti-inflammatory properties. However, these elements havenot been previously identified. We have now established a new family ofhuman Serpin-derived peptides with potent anti-inflammatory effectusing, e.g., a mouse endotoxemia model (LPS induced endotoxemia). Basedon the efficacy of the peptides in the mouse inflammation model, thepeptide size, and the safety profile of the parent protein, theAAT-based peptides, the peptides, such as SP16, and fragments andderivatives thereof provide a novel and improved molecule to treatinflammation in humans.

Formula I provides a composition comprising a peptide comprising,consisting essentially of or consisting of the amino acid sequenceX1-Z1-F-N-R-P-F-X2-Z2-X3-Z3-Q (SEQ ID NO:35) andX1-Z1-F-N-K-P-F-X2-Z2-X3-Z3 (SEQ ID NO: 2) wherein

X1 is V or L;

X2 is V, L or M;

X3 is M, I or V;

Z1 is any amino acid;

Z2 is a sequence of any two amino acids; and

Z3 is a sequence any five amino acids, wherein the peptide comprises 37or fewer amino acids.

In certain aspects of all the embodiments, the isolated peptide causes a50% or 75% decrease in serum TNF-α levels compared to the serum level asmeasured prior to administering the isolated peptide, when administeredin an effective amount to a human subject. In some aspects of all theembodiments of the invention, the peptide further comprises a fusionprotein. The fusion protein can be selected from an epitope tag and ahalf-life extender or a combination thereof.

In certain aspects of all the embodiments, the isolated peptide causesimprovement in glycemic control in diabetic subjects, as measured using,e.g., A1C test, fasting plasma glucose test (FPG), and/or the oralglucose tolerance test (OGTT). Pre-diabetic individuals typically scoreat or above 5.7% to under 6.5% on the A1C test, whereas diabetics scoreover 6.5% on this test. Pre-diabetic individuals also typically score ator over 100 mg/dl to under 126 mg/dl using the FPG test whereasdiabetics score over 126 mg/dl. Pre-diabetic individuals typically scoreat or over 140 to under 200 mg/dl using the OGTT test whereas diabeticindividuals score over 200 mg/dl.

Formula II provides a composition comprising an isolated peptidecomprising, consisting essentially of or consisting of the amino acidsequence X1-Z1-F-N-X2-P-F-X3-Z2-X4-Z3-X5 (SEQ ID NO: 3), wherein

X1 is V or L;

X2 is K or R;

X3 is V, L or M;

X4 is M, I or V;

X5 is K or Q;

Z1 is any amino acid;

Z2 is a sequence of any two amino acids;

Z3 is a sequence any five amino acids; and wherein the isolated peptidecauses a 75% decrease in serum TNF-α levels compared to the serum levelsprior to administering the isolated peptide when administered in aneffective amount to a human subject.

In certain embodiments, the peptide comprising the amino acid sequenceof X1-Z1-F-N-X2-P-F-X3-Z2-X4-Z3-X5 (SEQ ID NO: 3) as defined above,includes, at most, 35, 22 or 21 amino acid residues. In certain aspectsof all the embodiments of the invention, the peptide further comprises afusion protein. Specifically the fusion protein can be selected from anepitope tag and a half-life extender or a combination thereof.

In some aspects of all the methods and uses of the invention, thepeptide is SP16.

Therefore, the invention also provides an isolated peptide consisting ofor consisting essentially of the amino acid sequence RFNRPFLR (SEQ IDNO: 4) and RFNKPFLR (SEQ ID NO: 5), which can also be used for thetreatment of inflammation. In certain embodiments, the peptide causes a50% or 75% decrease in serum TNF-α levels compared to the serum TNF-αlevels prior to administering the isolated peptide when administered inan effective amount to a human subject. In some aspects of all theembodiments of the invention, the isolated peptide further comprises afusion protein. Specifically the fusion protein can be selected from anepitope tag and a half-life extender. In other embodiments, the isolatedpeptide comprises, at most, 100, 35, 22, 21, 16 or 9 amino. In otherembodiments, the isolated peptide comprises the amino acid sequence ofZ1-RFNRPFLR-Z2 (SEQ ID NO: 6), and Z1-RFNKPFLR-Z2 (SEQ ID NO: 7) whereinZ1 and Z2 are independently between 1, 2, 3, 4, 5, 6, 6, 7, 8, 9, 10 orbetween 1 and 3, between 1 and 5, between 1 and 6, between 1 and 7,between 1 and 8, between 1 and 9, or between 1 and 10 basic amino acids.

In some aspects of all the embodiments of the invention, the isolatedpeptide consists essentially of or consists of the amino acid sequenceof RRRFNRPFLRRR (SEQ ID NO: 8) and RRRFNKPFLRRR (SEQ ID NO: 9). Thedisclosure also provides a composition comprising a peptide consistingessentially of or consisting of the amino acid sequence of FNRPFL (SEQID NO: 10) and FNKPFL (SEQ ID NO: 11).

In certain embodiments the isolated peptide comprises 5 or moresequential amino acids from the amino acid sequence of FLMIEQNTK (SEQ IDNO: 36). These peptides can be used to reduce inflammation or reduceTNF-α level in a subject. In certain embodiments, the amount of TNF-αlevel in the serum is reduced compared to the amount of TNF-α in theserum prior to administering the isolated peptide. In certainembodiments, the glycemic control is improved as measured using testssuch as A1C test, fasting plasma glucose test (FPG), and the oralglucose tolerance test (OGTT). Also, both TNF-α level and glycemiccontrol can be used when measuring improvement in a diabetic individual.

In some aspects, the method of treatment of inflammation furthercomprises analysis of TNF-α serum levels prior to administering theisolated peptide and after administering the isolated peptide. If theTNF-α serum level is decreased less than 30%, the step of administeringcan be repeated with the same dose or with a larger dose of the peptidecompared to the first dose.

Fragments of any of the peptides described above can vary in size. Forexample, these fragments can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, or 37, amino acids in length.

The peptides described above are generally used to reduce inflammation.The peptides exert anti-inflammatory and immune-modulating effects, andadditionally, directly or indirectly stimulate beta cell regeneration.In certain embodiments, these peptides reduce inflammation by reducingthe activity or expression of TNF-α. The activity of TNF-α can bereduced by 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, 99 or 100%. The expression of TNF-α can be reduced 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 or100%. When administered therapeutically, the peptide compositiontypically further comprises a pharmaceutically acceptable solution orcarrier.

The peptides described above can also be used to treat, prevent orimprove the symptoms of several pathologies. These pathologies includeinflammation, auto-immune tissue destruction and hyperglycemia. Thepeptides described above can also be used to stimulate expansion of betacell mass in an individual with diabetes, treatment of cystic fibrosis,and in treatment or prevention of endotoxemia following acute radiationexposure and in burn patients.

Accordingly, the disclosure further provides methods of treatinginflammation comprising the step of administering any one of thepeptides described herein or a combination thereof to a subject in needof treatment of inflammation. In some aspects, the subject has not beentreated with alpha-antitrypsin prior to the treatment. In some aspects,the method comprises a step of assaying whether the individual hasincreased serum TNF-α levels and if the subject has increased serumTNF-α levels then administering the peptide to the subject, and if not,then not administering the peptide to the subject.

The disclosure also enables methods of preventing development ofinflammation comprising the step of administering any one of thepeptides or a combination thereof to a subject in need of prevention ofinflammation. In some aspects, the subject has not been treated withalpha-antitrypsin prior to the treatment. In some aspects, the methodcomprises a step of assaying whether the individual has increased serumTNF-α levels and if the subject has increased serum TNF-α levels thenadministering the peptide to the subject, and if not, then notadministering the peptide to the subject.

The disclosure also enables methods of preventing autoimmune tissuedestruction comprising the step of administering any one of the peptidesor a combination thereof to a subject in need of prevention ofautoimmune tissue destruction. In some aspects, the subject has not beentreated with alpha-antitrypsin prior to the treatment. In some aspects,the method comprises a step of assaying whether the individual hasincreased serum TNF-α levels and if the subject has increased serumTNF-α levels then administering the peptide to the subject, and if not,then not administering the peptide to the subject.

The disclosure also enables methods of improving glycemic control inindividuals having diabetes comprising the step of administering any oneof the peptides or a combination thereof to a subject in need ofprevention of autoimmune tissue destruction. In some aspects, thesubject has not been treated with alpha-antitrypsin prior to thetreatment. In some aspects, the method comprises a step of assayingwhether the individual has improved glycemic control by e.g., A1C test,fasting plasma glucose test, and/or the oral glucose tolerance test orany other known test for measuring the functioning of glycemic control.

In one embodiment, the disclosure provides use of any one or acombination of the isolated or synthesized peptides for the treatment ofinflammation, and inflammation wherein the TNF-α level is increased.

In certain aspects of all the embodiments, inflammation is related todiabetes type 1 or 2, rheumatoid arthritis or COPD.

For example, the Examples provided herein, demonstrate that the peptidesdisclosed herein are efficacious in treating both type I and type IIdiabetes. For example, when SP16 is administered to db/db mice, arecognized type II diabetes model animal, non-fasted blood glucose andHbA1c levels were lowered, while serum c-peptide levels increased, andglucose tolerance was improved in response to the peptides (see, e.g.,FIG. 8A-8F). This demonstrates that the peptide described hereinimproves glycemic control in type II diabetes. As a further example,when NOD mice, a recognized model of type I diabetes were administeredwith peptides as described herein, the development of type I diabeteswas delayed and/or prevented as demonstrated in FIG. 18. The Examplesinclude peptide administration by intraperitoneal injection as well asby oral gavage, demonstrating that oral administration is suitable fordelivering the peptides of the invention. Without wishing to be bound bya theory, we suggest that, the peptides, such as SP16, work by oraladministration via the gut associated lymphoid tissue (GALT).

Again, without wishing to the bound by a theory, we suggest that TLR2agonism or TLR4 signaling by the peptides disclosed herein, such asSP16, can be used to induce expansion of gut Tregs, enabling oralimmunotherapy using the peptides of the invention.

The invention also provides methods for treatment and prevention ofcystic fibrosis and endotoxemia following acute radiation exposure andin burn patients using any one or a combination of the peptides of theinvention.

For convenience, certain terms employed in the entire application(including the specification, examples, and appended claims) are definedthroughout the specification. Unless defined otherwise, all technicaland scientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs.

The term “wild type” refers to the naturally-occurring polynucleotidesequence encoding a protein, or a portion thereof, or protein sequence,or portion thereof, respectively, as it normally exists in vivo.

The term “mutant” refers to any change in the genetic material of anorganism, in particular a change (i.e., deletion, substitution,addition, or alteration) in a wild-type polynucleotide sequence or anychange in a wild-type protein sequence. Although it is often assumedthat a change in the genetic material results in a change of thefunction of the protein, the term—“mutant” refers to a change in thesequence of a wild-type protein regardless of whether that change altersthe function of the protein (e.g., increases, decreases, imparts a newfunction), or whether that change has no effect on the function of theprotein (e.g., the mutation or variation is silent). The term mutationis used interchangeably herein with polymorphism in this application.

The terms “polypeptide” and “protein” are used interchangeably to referto an isolated polymer of amino acid residues, and are not limited to aminimum length unless otherwise defined. Peptides, oligopeptides,dimers, multimers, and the like, are also composed of linearly arrangedamino acids linked by peptide bonds, and whether produced biologicallyand isolated from the natural environment, produced using recombinanttechnology, or produced synthetically typically using naturallyoccurring amino acids.

In some aspects, the polypeptide or protein is a “modified polypeptide”comprising non-naturally occurring amino acids. In some aspects, thepolypeptides comprise a combination of naturally occurring andnon-naturally occurring amino acids, and in some embodiments, thepeptides comprise only non-naturally occurring amino acids.

In some aspects of all the embodiments of the invention, the peptides ormodified peptides further comprise co-translational andpost-translational (C-terminal peptide cleavage) modifications, such as,for example, disulfide-bond formation, glycosylation, acetylation,phosphorylation, proteolytic cleavage (e.g., cleavage by furins ormetalloproteases), and the like to the extent that such modifications donot affect the anti-inflammatory properties of the isolated peptides ortheir capacity to improve glycemic control.

In some aspects of the invention, the polypeptide is altered. The term“altered polypeptide” refers to a peptide that includes alterations,such as deletions, additions, and substitutions (generally conservativein nature as would be known to a person in the art, such as alanines),to the native sequence, as long as the protein maintains the desiredactivity, i.e., it anti-inflammatory activity of capacity to improveglycemic control or reduce hyperglycemia. These modifications can bedeliberate, as through site-directed mutagenesis, or can be accidental,such as through mutations of artificial hosts, such as geneticallyengineered bacteria, yeast or mammalian cells, that produce theproteins, or errors due to PCR amplification or other recombinant DNAmethods. Polypeptides or proteins are composed of linearly arrangedamino acids linked by peptide bonds, but in contrast to peptides, have awell-defined conformation. Proteins, as opposed to peptides, generallyconsist of chains of 50 or more amino acids.

The term “peptide” as used herein typically refers to a sequence ofamino acids made up of a single chain of amino acids joined by peptidebonds. Generally, peptides contain at least two amino acid residues andare less than about 50 amino acids in length, unless otherwise defined.

“Modified peptide” may include the incorporation of non-natural aminoacids into the peptides of the invention, including synthetic non-nativeamino acids, substituted amino acids, or one or more D-amino acids intothe peptides (or other components of the composition, with exception forprotease recognition sequences) is desirable in certain situations.D-amino acid-containing peptides exhibit increased stability in vitro orin vivo compared to L-amino acid-containing forms. Thus, theconstruction of peptides incorporating D-amino acids can be particularlyuseful when greater in vivo or intracellular stability is desired orrequired. More specifically, D-peptides are resistant to endogenouspeptidases and proteases, thereby providing better oral trans-epithelialand transdermal delivery of linked drugs and conjugates, improvedbioavailability of membrane-permanent complexes (see below for furtherdiscussion), and prolonged intravascular and interstitial lifetimes whensuch properties are desirable. The use of D-isomer peptides can alsoenhance transdermal and oral trans-epithelial delivery of linked drugsand other cargo molecules. Additionally, D-peptides cannot be processedefficiently for major histocompatibility complex class II-restrictedpresentation to T helper cells, and are therefore less likely to inducehumoral immune responses in the whole organism. Peptide conjugates cantherefore be constructed using, for example, D-isomer forms of cellpenetrating peptide sequences, L-isomer forms of cleavage sites, andD-isomer forms of therapeutic peptides. Therefore, in some embodimentsthe peptides as disclosed comprise L and D amino acids, wherein no morethan 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 D-amino acids are included. Incertain aspects, the peptides comprise more than 10 D-amino acids, andin certain aspects all the amino acids of the peptides are D-aminoacids.

In yet a further aspect, the peptides or fragments or derivativesthereof can be “retro-inverso peptides.” A “retro-inverso peptide”refers to a peptide with a reversal of the direction of the peptide bondon at least one position, i.e., a reversal of the amino- andcarboxy-termini with respect to the side chain of the amino acid. Thus,a retro-inverso analogue has reversed termini and reversed direction ofpeptide bonds while approximately maintaining the topology of the sidechains as in the native peptide sequence. The retro-inverso peptide cancontain L-amino acids or D-amino acids, or a mixture of L-amino acidsand D-amino acids, up to all of the amino acids being the D-isomer.Partial retro-inverso peptide analogues are polypeptides in which onlypart of the sequence is reversed and replaced with enantiomeric aminoacid residues. Since the retro-inverted portion of such an analogue hasreversed amino and carboxyl termini, the amino acid residues flankingthe retro-inverted portion are replaced by side-chain-analogousa-substituted geminal-diaminomethanes and malonates, respectively.Retro-inverso forms of cell penetrating peptides have been found to workas efficiently in translocating across a membrane as the natural forms.Synthesis of retro-inverso peptide analogues are described in Bonelli,F. et al., Int J Pept Protein Res. 24(6):553-6 (1984); Verdini, A andViscomi, G. C, J. Chem. Soc. Perkin Trans. 1:697-701 (1985); and U.S.Pat. No. 6,261,569, which are incorporated herein in their entirety byreference. Processes for the solid-phase synthesis of partialretro-inverso peptide analogues have been described (EP 97994-B) whichis also incorporated herein in its entirety by reference.

The terms “homology”, “identity” and “similarity” refer to the degree ofsequence similarity between two peptides or between two optimallyaligned nucleic acid molecules. Homology and identity can each bedetermined by comparing a position in each sequence which can be alignedfor purposes of comparison. For example, it is based upon using standardhomology software in the default position, such as BLAST, version2.2.14. When an equivalent position in the compared sequences isoccupied by the same base or amino acid, then the molecules areidentical at that position; when the equivalent site occupied by similaramino acid residues (e.g., similar in steric and/or electronic naturesuch as, for example conservative amino acid substitutions), then themolecules can be referred to as homologous (similar) at that position.Expression as a percentage of homology/similarity or identity refers toa function of the number of similar or identical amino acids atpositions shared by the compared sequences, respectfully. A sequencewhich is “unrelated” or “non-homologous” shares less than 40% identity,though preferably less than 25% identity with the sequences as disclosedherein.

As used herein, the term “sequence identity” means that twopolynucleotide or amino acid sequences are identical (i.e., on anucleotide-by-nucleotide or residue-by-residue basis) over thecomparison window. The term “percentage of sequence identity” iscalculated by comparing two optimally aligned sequences over the windowof comparison, determining the number of positions at which theidentical nucleic acid base (e.g., A, T. C, G. U. or I) or residueoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the comparison window (i.e., the window size), andmultiplying the result by 100 to yield the percentage of sequenceidentity.

The term “substantial identity” as used herein denotes a characteristicof a polynucleotide or amino acid sequence, wherein the polynucleotideor amino acid comprises a sequence that has at least 85% sequenceidentity, preferably at least 90% to 95% sequence identity, more usuallyat least 99% sequence identity as compared to a reference sequence overa comparison window of at least 18 nucleotide (6 amino acid) positions,frequently over a window of at least 24-48 nucleotide (8-16 amino acid)positions, wherein the percentage of sequence identity is calculated bycomparing the reference sequence to the sequence which can includedeletions or additions which total 20 percent or less of the referencesequence over the comparison window. The reference sequence can be asubset of a larger sequence. The term “similarity”, when used todescribe a polypeptide, is determined by comparing the amino acidsequence and the conserved amino acid substitutes of one polypeptide tothe sequence of a second polypeptide.

As used herein, the terms “homologous” or “homologues” are usedinterchangeably, and when used to describe a polynucleotide orpolypeptide, indicates that two polynucleotides or polypeptides, ordesignated sequences thereof, when optimally aligned and compared, forexample using BLAST, version 2.2.14 with default parameters for analignment (see herein) are identical, with appropriate nucleotideinsertions or deletions or amino-acid insertions or deletions, in atleast 70% of the nucleotides, usually from about 75% to 99%, and morepreferably at least about 98 to 99% of the nucleotides. The term“homolog” or “homologous” as used herein also refers to homology withrespect to structure and/or function. With respect to sequence homology,sequences are homologs if they are at least 50%, at least 60 at least70%, at least 80%, at least 90%, at least 95% identical, at least 97%identical, or at least 99% identical. Determination of homologs of thegenes or peptides of the present invention can be easily ascertained bythe skilled artisan.

The term “substantially homologous” refers to sequences that are atleast 90%, at least 95% identical, at least 96%, identical at least 97%identical, at least 98% identical or at least 99% identical. Homologoussequences can be the same functional gene in different species.Determination of homologs of the genes or peptides of the presentinvention can be easily ascertained by the skilled artisan.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, forexample, by the local homology algorithm of Smith and Waterman (Adv.Appl. Math. 2:482 (1981), which is incorporated by reference herein), bythe homology alignment algorithm of Needleman and Wunsch (J. MoI. Biol.48:443-53 (1970), which is incorporated by reference herein), by thesearch for similarity method of Pearson and Lipman (Proc. Natl. Acad.Sci. USA 85:2444-48 (1988), which is incorporated by reference herein),by computerized implementations of these algorithms (e.g., GAP, BESTFIT,FASTA, and TFASTA in the Wisconsin Genetics Software Package, GeneticsComputer Group, 575 Science Dr., Madison, Wis.), or by visualinspection. (See generally Ausubel et al. (eds.), Current Protocols inMolecular Biology, 4th ed., John Wiley and Sons, New York (1999)).

One example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments to show the percent sequence identity. It also plotsa tree or dendogram showing the clustering relationships used to createthe alignment. PILEUP uses a simplification of the progressive alignmentmethod of Feng and Doolittle (J. MoI. Evol. 25:351-60 (1987), which isincorporated by reference herein). The method used is similar to themethod described by Higgins and Sharp (Comput. Appl. Biosci. 5:151-53(1989), which is incorporated by reference herein). The program canalign up to 300 sequences, each of a maximum length of 5,000 nucleotidesor amino acids. The multiple alignment procedure begins with thepairwise alignment of the two most similar sequences, producing acluster of two aligned sequences. This cluster is then aligned to thenext most related sequence or cluster of aligned sequences. Two clustersof sequences are aligned by a simple extension of the pairwise alignmentof two individual sequences. The final alignment is achieved by a seriesof progressive, pairwise alignments. The program is run by designatingspecific sequences and their amino acid or nucleotide coordinates forregions of sequence comparison and by designating the programparameters. For example, a reference sequence can be compared to othertest sequences to determine the percent sequence identity relationshipusing the following parameters: default gap weight (3.00), default gaplength weight (0.10), and weighted end gaps.

Another example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described by Altschul et al. (J. MoI. Biol. 215:403-410 (1990), whichis incorporated by reference herein). (See also Zhang et al., NucleicAcid Res. 26:3986-90 (1998); Altschul et al., Nucleic Acid Res.25:3389-402 (1997), which are incorporated by reference herein).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information internet web site. Thisalgorithm involves first identifying high scoring sequence pairs (HSPs)by identifying short words of length W in the query sequence, whicheither match or satisfy some positive-valued threshold score T whenaligned with a word of the same length in a database sequence. T isreferred to as the neighborhood word score threshold (Altschul et al.(1990), supra). These initial neighborhood word hits act as seeds forinitiating searches to find longer HSPs containing them. The word hitsare then extended in both directions along each sequence for as far asthe cumulative alignment score can be increased. Extension of the wordhits in each direction is halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T, and Xdetermine the sensitivity and speed of the alignment. The BLAST programuses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix(see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-9(1992), which is incorporated by reference herein) alignments (B) of 50,expectation (E) of 10, M=5, N=−4, and a comparison of both strands.

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci.USA 90:5873-77 (1993), which is incorporated by reference herein). Onemeasure of similarity provided by the BLAST algorithm is the smallestsum probability (P(N)), which provides an indication of the probabilityby which a match between two nucleotide or amino acid sequences wouldoccur by chance. For example, an amino acid sequence is consideredsimilar to a reference amino acid sequence if the smallest sumprobability in a comparison of the test amino acid to the referenceamino acid is less than about 0.1, more typically less than about 0.01,and most typically less than about 0.001.

The term “variant” as used herein refers to a peptide or nucleic acidthat differs from the polypeptide or nucleic acid by one or more aminoacid or nucleic acid deletions, additions, substitutions or side-chainmodifications, yet retains one or more specific functions or biologicalactivities of the naturally occurring molecule. Amino acid substitutionsinclude alterations in which an amino acid is replaced with a differentnaturally-occurring or a non-conventional amino acid residue. Suchsubstitutions may be classified as “conservative”, in which case anamino acid residue contained in a polypeptide is replaced with anothernaturally occurring amino acid of similar character either in relationto polarity, side chain functionality or size. Such conservativesubstitutions are well known in the art. Substitutions encompassed bythe present invention may also be “non-conservative”, in which an aminoacid residue which is present in a peptide is substituted with an aminoacid having different properties, such as naturally-occurring amino acidfrom a different group (e.g., substituting a charged or hydrophobicamino; acid with alanine), or alternatively, in which anaturally-occurring amino acid is substituted with a non-conventionalamino acid. In some embodiments amino acid substitutions areconservative. Also encompassed within the term variant when used withreference to a polynucleotide or polypeptide, refers to a polynucleotideor polypeptide that can vary in primary, secondary, or tertiarystructure, as compared to a reference polynucleotide or polypeptide,respectively (e.g., as compared to a wild-type polynucleotide orpolypeptide).

Variants can also be synthetic, recombinant, or chemically modifiedpolynucleotides or polypeptides isolated or generated using methods wellknown in the art. Variants can include conservative or non-conservativeamino acid changes, as described below. Polynucleotide changes canresult in amino acid substitutions, additions, deletions, fusions andtruncations in the polypeptide encoded by the reference sequence.Variants can also include insertions, deletions or substitutions ofamino acids, including insertions and substitutions of amino acids andother molecules) that do not normally occur in the peptide sequence thatis the basis of the variant, for example but not limited to insertion ofornithine which do not normally occur in human proteins. The term“conservative substitution,” when describing a polypeptide, refers to achange in the amino acid composition of the polypeptide that does notsubstantially alter the polypeptide's activity. For example, aconservative substitution refers to substituting an amino acid residuefor a different amino acid residue that has similar chemical properties.Conservative amino acid substitutions include replacement of a leucinewith an isoleucine or valine, an aspartate with a glutamate, or athreonine with a serine.

“Conservative amino acid substitutions” result from replacing one aminoacid with another having similar structural and/or chemical properties,such as the replacement of a leucine with an isoleucine or valine, anaspartate with a glutamate, or a threonine with a serine. Thus, a“conservative substitution” of a particular amino acid sequence refersto substitution of those amino acids that are not critical forpolypeptide activity or substitution of amino acids with other aminoacids having similar properties (e.g., acidic, basic, positively ornegatively charged, polar or non-polar, etc.) such that the substitutionof even critical amino acids does not reduce the activity of thepeptide, (i.e. the ability of the peptide to penetrate the blood brainbarrier (BBB)). Conservative substitution tables providing functionallysimilar amino acids are well known in the art. For example, thefollowing six groups each contain amino acids that are conservativesubstitutions for one another: 1) Alanine (A), Serine (S), Threonine(T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N),Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine(L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W). (See also Creighton, Proteins, W. H. Freeman and Company(1984), incorporated by reference in its entirety.) In some embodiments,individual substitutions, deletions or additions that alter, add ordelete a single amino acid or a small percentage of amino acids can alsobe considered “conservative substitutions” if the change does not reducethe activity of the peptide. Insertions or deletions are typically inthe range of about 1 to 5 amino acids. The choice of conservative aminoacids may be selected based on the location of the amino acid to besubstituted in the peptide, for example if the amino acid is on theexterior of the peptide and expose to solvents, or on the interior andnot exposed to solvents.

In alternative embodiments, one can select the amino acid which willsubstitute an existing amino acid based on the location of the existingamino acid, i.e. its exposure to solvents (i.e. if the amino acid isexposed to solvents or is present on the outer surface of the peptide orpolypeptide as compared to internally localized amino acids not exposedto solvents). Selection of such conservative amino acid substitutionsare well known in the art, for example as disclosed in Dordo et al, J.MoI Biol, 1999, 217, 721-739 and Taylor et al, J. Theor. Biol.119(1986);205-218 and S. French and B. Robson, J. MoI. Evol.19(1983)171. Accordingly, one can select conservative amino acidsubstitutions suitable for amino acids on the exterior of a protein orpeptide (i.e. amino acids exposed to a solvent), for example, but notlimited to, the following substitutions can be used: substitution of Ywith F, T with S or K, P with A, E with D or Q, N with D or G, R with K,G with N or A, T with S or K, D with N or E, I with L or V, F with Y, Swith T or A, R with K, G with N or A, K with R, A with S, K or P.

In alternative embodiments, one can also select conservative amino acidsubstitutions encompassed suitable for amino acids on the interior of aprotein or peptide, for example one can use suitable conservativesubstitutions for amino acids is on the interior of a protein or peptide(i.e. the amino acids are not exposed to a solvent), for example but notlimited to, one can use the following conservative substitutions: whereY is substituted with F, T with A or S, I with L or V, W with Y, M withL, N with D, G with A, T with A or S, D with N, I with L or V, F with Yor L, S with A or T and A with S, G, T or V. In some embodiments,non-conservative amino acid substitutions are also encompassed withinthe term of variants.

The term “derivative” as used herein refers to peptides which have beenchemically modified, for example but not limited to by techniques suchas ubiquitination, labeling, pegylation (derivatization withpolyethylene glycol), lipidation, glycosylation, or addition of othermolecules. A molecule also a “derivative” of another molecule when itcontains additional chemical moieties not normally a part of themolecule. Such moieties can improve the molecule's solubility,absorption, biological half-life, etc. The moieties can alternativelydecrease the toxicity of the molecule, eliminate or attenuate anyundesirable side effect of the molecule, etc. Moieties capable ofmediating such effects are disclosed in Remington's PharmaceuticalSciences, 18th edition, A. R. Gennaro, Ed., MackPubl., Easton, Pa.(1990), incorporated herein, by reference, in its entirety.

Thus, in certain aspects of all the embodiments of the invention, thepeptides of the invention comprise peptide derivatives, such aspegylated peptides.

The term “functional” when used in conjunction with “derivative” or“variant” refers to a peptide of the invention which possesses abiological activity (either functional or structural) that issubstantially similar to a biological activity of the entity or moleculeit is a functional derivative or functional variant thereof, i.e.,anti-inflammatory activity in the context of the peptides describedherein. The term functional derivative is intended to include thefragments, analogues or chemical derivatives of a molecule.

The term “insertions” or “deletions” are typically in the range of about1 to 5 amino acids. The variation allowed can be experimentallydetermined by producing the peptide synthetically while systematicallymaking insertions, deletions, or substitutions of nucleotides in thesequence using recombinant DNA techniques.

The term “substitution” when referring to a peptide, refers to a changein an amino acid for a different entity, for example another amino acidor amino-acid moiety. Substitutions can be conservative ornon-conservative substitutions.

An “analog” of a molecule such as a peptide refers to a molecule similarin function to either the entire molecule or to a fragment thereof. Theterm “analog” is also intended to include allelic species and inducedvariants. Analogs typically differ from naturally occurring peptides atone or a few positions, often by virtue of conservative substitutions.Analogs typically exhibit at least 80 or 90% sequence identity withnatural peptides. Some analogs also include unnatural amino acids ormodifications of N or C terminal amino acids. Examples of unnaturalamino acids are, for example but not limited to; disubstituted aminoacids, N-alkyl amino acids, lactic acid, 4-hydroxyproline,γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine,O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine,5-hydroxylysine, σ-N-methylarginine. Fragments and analogs can bescreened for prophylactic or therapeutic efficacy in transgenic animalmodels as described below.

By “covalently bonded” is meant joined either directly or indirectly(e.g., through a linker) by a covalent chemical bond. In some aspects ofall the embodiments of the invention, the fusion peptides are covalentlybonded.

The term “fusion protein” as used herein refers to a recombinant proteinof two or more proteins. Fusion proteins can be produced, for example,by a nucleic acid sequence encoding one protein is joined to the nucleicacid encoding another protein such that they constitute a singleopen-reading frame that can be translated in the cells into a singlepolypeptide harboring all the intended proteins. The order ofarrangement of the proteins can vary. Fusion proteins can include anepitope tag or a half-life extender. Epitope tags include biotin, FLAGtag, c-myc, hemaglutinin, His6 (SEQ ID NO: 37), digoxigenin, FITC, Cy3,Cy5, green fluorescent protein, V5 epitope tags, GST, β-galactosidase,AU1, AU5, and avidin. Half-life extenders include Fc domain and serumalbumin.

The terms “subject” and “individual” and “patient” are usedinterchangeably herein, and refer to an animal, for example a human ornon-human animal (e.g., a mammal), to whom treatment, includingprophylactic treatment, with a pharmaceutical composition as disclosedherein, is provided. The term “subject” as used herein refers to humanand non-human animals. The term “non-human animals” includes allvertebrates, e.g., mammals, such as non-human primates, (particularlyhigher primates), sheep, dogs, rodents (e.g. mouse or rat), guinea pigs,goats, pigs, cats, rabbits, cows, and non-mammals such as chickens,amphibians, reptiles etc. In one embodiment, the subject is human. Inanother embodiment, the subject is an experimental animal or animalsubstitute as a disease model. Non-human mammals include mammals such asnon-human primates, (particularly higher primates), sheep, dogs, rodents(e.g. mouse or rat), guinea pigs, goats, pigs, cats, rabbits and cows.In some aspects, the non-human animal is a companion animal such as adog or a cat.

“Treating” a disease or condition in a subject or “treating” a patienthaving a disease or condition refers to subjecting the individual to apharmaceutical treatment, e.g., the administration of a drug, such thatat least one symptom of the disease or condition is decreased orstabilized. Typically, when the peptide is administered therapeuticallyas a treatment, it is administered to a subject who presents with one ormore symptoms of inflammation.

The term “prevention” is used in connection of prevention of symptoms orslowing down of symptom development from the time of asymptomatic state.Typically, when the peptide is administered preventively, it isadministered to a subject who does not present imminent symptoms ofinflammation. Typically, the subject is at risk of developinginflammation, such as diabetes or COPD, due to the family history,laboratory results, genetic testing or life-style. In some aspects, the“prevention” relates to administering the peptides to burn victims orpeople that have been subjected to acute radiation exposure prior tothem developing endotoxemia because these subjects are at risk ofdeveloping endotoxemia as a result of tissue damage caused by a burn orradiation.

By “specifically binds” or “specific binding” is meant a compound orantibody that recognizes and binds a desired polypeptide but that doesnot substantially recognize and bind other molecules in a sample, forexample, a biological sample, which naturally includes a polypeptide ofthe invention. Specific binding can be characterized by a dissociationconstant of at least about 1×10−6 M or smaller. In other embodiments,the dissociation constant is at least about 1×10−7 M, 1×10−8 M, or1×10−9 M. Methods for determining whether two molecules specificallybind are well known in the art and include, for example, equilibriumdialysis, surface plasmon resonance, and the like.

By “isolated” it is meant that the polypeptide has been separated fromany natural environment, such as a body fluid, e.g., blood, andseparated from the components that naturally accompany the peptide.

By isolated and “substantially pure” is meant a polypeptide that hasbeen separated and purified to at least some degree from the componentsthat naturally accompany it. Typically, a polypeptide is substantiallypure when it is at least about 60%, or at least about 70%, at leastabout 80%, at least about 90%, at least about 95%, or even at leastabout 99%, by weight, free from the proteins and naturally-occurringorganic molecules with which it is naturally associated. For example, asubstantially pure polypeptide may be obtained by extraction from anatural source, by expression of a recombinant nucleic acid in a cellthat does not normally express that protein, or by chemical synthesis.

By a “decrease” or “inhibition” used in the context of the level of, forexample TNF-alpha levels refers to reduction of the amount of protein inthe biological sample, such as blood or tissue sample, a cell, a cellextract, or a cell supernatant. For example, such a decrease may be dueto reduced RNA stability, transcription, or translation, increasedprotein degradation, or RNA interference. Preferably, this decrease isat least about 5%, at least about 10%, at least about 25%, at leastabout 50%, at least about 75%, at least about 80%, or even at leastabout 90% compared to a reference value.

The term “reference value” in the context of the claims and theapplication refers typically to an abnormally high TNF-alpha level foundin an individual affected with or suffering from inflammation. Thereference value is typically the amount of TNF-alpha in the individualprior to administering of the peptide of the invention. In some aspectsof all the embodiments concerning glycemic control, the term “referencevalue” refers to the numeric values used in measuring glycemic controlin a subject. There are a number of tests which can be used to determineif, e.g., a human subject is affected with pre-diabetes. Such testsinclude, e.g., the A1C test, fasting plasma glucose test (FPG), and theoral glucose tolerance test (OGTT). Examples of reference values usingthese methods follow: Pre-diabetic individuals typically score at orabove 5.7% to under 6.5% on the A1C test, whereas diabetics score over6.5% on this test. Pre-diabetic individuals also typically score at orover 100 mg/dl to under 126 mg/dl using the FPG test whereas diabeticsscore over 126 mg/dl. Pre-diabetic individuals typically score at orover 140 to under 200 mg/dl using the OGTT test whereas diabeticindividuals score over 200 mg/dl. Thus, referring to a normoglycemicreference value, one can use the following cut-off numbers whenmeasuring is performed using the above-mentioned tests: A1C test—under5.7%; FPG test under 100 mg/dl; and OGTT under 140 mg/dl.

By an “increase” in the expression or activity of a gene or protein ismeant a positive change in protein or nucleic acid level or activity ina cell, a cell extract, or a cell supernatant. For example, such anincrease may be due to increased RNA stability, transcription, ortranslation, or decreased protein degradation. Preferably, this increaseis at least 5%, at least about 10%, at least about 25%, at least about50%, at least about 75%, at least about 80%, at least about 100%, atleast about 200%, or even about 500% or more over the level ofexpression or activity under control conditions.

The term “recombinant” as used herein to describe a nucleic acidmolecule, means a polynucleotide of genomic, cDNA, viral, semisynthetic,and/or synthetic origin, which, by virtue of its origin or manipulation,is not associated with all or a portion of the polynucleotide with whichit is associated in nature. The term recombinant as used with respect toa protein or polypeptide, means a polypeptide produced by expression ofa recombinant polynucleotide. The term recombinant as used with respectto a host cell means a host cell into which a recombinant polynucleotidehas been introduced. Recombinant is also used herein to refer to, withreference to material (e.g., a cell, a nucleic acid, a protein, or avector) that the material has been modified by the introduction of aheterologous material (e.g., a cell, a nucleic acid, a protein, or avector).

The term “vectors” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked; a plasmidis a species of the genus encompassed by “vector”. The term “vector”typically refers to a nucleic acid sequence containing an origin ofreplication and other entities necessary for replication and/ormaintenance in a host cell. Vectors capable of directing the expressionof genes and/or nucleic acid sequence to which they are operativelylinked are referred to herein as “expression vectors”. In general,expression vectors of utility are often in the form of “plasmids” whichrefer to circular double stranded DNA loops which, in their vector formare not bound to the chromosome, and typically comprise entities forstable or transient expression or the encoded DNA. Other expressionvectors can be used in the methods as disclosed herein for example, butare not limited to, plasmids, episomes, bacterial artificialchromosomes, yeast artificial chromosomes, bacteriophages or viralvectors, and such vectors can integrate into the host's genome orreplicate autonomously in the particular cell. A vector can be a DNA orRNA vector. Other forms of expression vectors known by those skilled inthe art which serve the equivalent functions can also be used, forexample self-replicating extrachromosomal vectors or vectors whichintegrates into a host genome. Preferred vectors are those capable ofautonomous replication and/or expression of nucleic acids to which theyare linked. Vectors capable of directing the expression of genes towhich they are operatively linked are referred to herein as “expressionvectors”.

The term “viral vectors” refers to the use of viruses, orvirus-associated vectors as carriers of a nucleic acid construct into acell. Constructs may be integrated and packaged into non-replicating,defective viral genomes like Adenovirus, Adeno-associated virus (AAV),or Herpes simplex virus (HSV) or others, including reteroviral andlentiviral vectors, for infection or transduction into cells. The vectormay or may not be incorporated into the cell's genome. The constructsmay include viral sequences for transfection, if desired. Alternatively,the construct may be incorporated into vectors capable of episomalreplication, e.g. EPV and EBV vectors.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., at least one) of the grammatical object of the article.By way of example, “an element” means one element or more than oneelement. Other than in the operating examples, or where otherwiseindicated, all numbers expressing quantities of ingredients or reactionconditions used herein should be understood as modified in all instancesby the term “about.” The term “about” when used in connection withpercentages can mean +1%. The present invention is further explained indetail by the following examples, but the scope of the invention shouldnot be limited thereto.

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such can vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention, which is defined solely by the claims.Other features and advantages of the invention will be apparent from thefollowing Detailed Description, the drawings, and the claims.

Treatment Methods of the Invention

One aspect of the present invention relates to the use of peptidesdescribed herein and mutants, variants, analogs or derivatives thereof.Specifically, these methods relate to administering any one of thepeptides as described herein or their pharmaceutically acceptablemodifications in a pharmaceutically acceptable carrier to a subject,e.g., a mammal in need thereof, e.g., a human, i.e., a subject havinginflammation or auto-immune tissue destruction or an individual withhyperglycemia or impaired glycemic control, such as an individual withtype 2 diabetes, and in need of protecting and/or stimulating expansionof beta cell mass, or an individual diagnosed with cystic fibrosis, oran individual at high risk of developing endotoxemia as a result of burnor acute radiation exposure. Also, we provide treatment of Rheumatoidarthritis, lupus and graft versus host disease, uveitis, eczema,psoriasis, IBD, and new onset type I diabetes.

Clinical descriptions of these diseases are well known. In some aspectsthe human is first diagnosed as having one or more symptom of thedisease before administering one or more of the peptides of theinvention. In some embodiments, the human has not previously beenadministered AAT as a treatment for the symptoms.

For example, we have shown in established preclinical mouse models forhuman diseases including type II diabetes (db/db), type I diabetes(NOD), rheumatoid arthritis (CAIA) and lethal endotoxemia that, forexample, the SP16 peptide significantly improves the symptoms.

We have also provided evidence that, e.g., the SP16 peptide can besafely administered using well-established preclinical safety studies.For example, we have also shown using FastPatch assay that, for example,the SP16 peptide does not impact hERG activity and we also were not ableto identify any hits on human receptor panning study (GenSEP Explorer)for the SP16 peptide.

The db/db mice carry a defective leptin receptor, which impairs theirability to regulate appetite and metabolism. The animals become obese at3-4 weeks of age, and initially show insulin resistance, which isfollowed by hyperglycemia at about 4-8 weeks of age. Severehyperglycemia is paralleled by depletion of the insulin-producingb-cells of the pancreatic islets and death by 10 months of age.

The db/db animals model the different phases of Type II Diabetes inhumans (FIG. 14). We have shown that the peptides of the inventionprovide benefits at early and late stages of disease. For example, wehave shown that SP16 improves glycemic control in the db/db model.

The blood glucose and HbA1c levels showed that SP16 treatment reduceshyperglycemia and improves glycemic control. In this study, five-weekold pre-diabetic db/db mice were assigned to groups of 10. The groupsreceived Saline (Vehicle control), o.6 mg/kg SP16 twice a week or 25mg/kg Rosiglitazone twice a week. Non-fasted blood glucose levels weredetermined twice a week using a glucometer. HbA1c levels were determinedusing the “A1cNow” monitor. Values represent averages for each group.(**) indicates p<0.01 and (*) indicates p<0.05 compared to the VehicleControl Group (T-test). See, FIG. 8A. It is also known that the targetnormalization value for human diabetes patients is also true for normalmouse, thus establishing the results from the mouse model directlyrelevant to human type II diabetes.

FIGS. 8B and 8D exemplify a graphs summarizing serum C-peptide levels(8B) and islet hyperplasia (8D) in the db/db mouse model of type IIdiabetes. Increased serum C-peptide levels are consistent with improvedβ-cell function in the treated groups. Five-week old pre-diabetic db/dbmice were assigned to groups of 10. Groups received Saline (Mock), 0.6mg/kg SP16 or 25 mg/kg Rosiglitazone twice a week. Pooled serumC-peptide levels were determined for each group . . . (*) indicatesp<0.05 and (**) p<0.01 compared to the Vehicle control.

We also showed that the SP16 lowered serum CRP levels when compared to avehicle control (FIG. 8E). Decreased CRP levels are consistent withreduced inflammation in the SP16 treated group of mice. Similarly,increased TGF-beta levels in SP16 treated mice are consistent with thepeptide treatment promoting an anti-inflammatory cytokine profile (FIG.8F). We assigned eight-week old diabetic db/db mice to 8 groups. Groupsreceived Saline (Mock) or 0.6 mg/kg SP16 biweekly for 12 weeks. Pooledserum CRP and TGF-beta levels were determined for each group. (*)indicates p<0.05 and (**) p<0.01 compared to the Vehicle control group.

The preclinical CAIA model of Rheumatoid Arthritis is a short studywhere arthritis is induced in Balb/c mice. On Day 0, animals areintravenously injected with a collagen antibody cocktail (MDBioSciences) which initiates autoimmune destruction of the collagen intheir joints. On Day 3, an intra-peritoneal injection of LPS is used toexacerbate the autoimmune reaction and inflammation. The readout in thismodel is paw swelling and histological assessment of joint erosion.Disease typically starts subsiding after 7-10 days.

We demonstrated that SP16 shows efficacy in the preclinical CAIA mousemodel. Specifically, we measured cumulative swelling scores for all pawsat the peak of disease (Day 7) for groups of 5 animals. Balb/c mice wereIV injected with a collagen antibody cocktail (MD BioSciences) on Day 0and IP injected with LPS on Day 3. Normal Control Animals received noinjections and served as disease-free baseline control. Daily SP16injection of 12 μg/day provided protection equivalent to Dexamethasoneand injections of 12 μg once every 3 days reduced the inflammation byalmost 50% compared to the treatment with the vehicle control. See e.g.,FIG. 9.

We also showed that SP16 lowers matrix metalloproteinase-1 (MMP-1)secretion in a cell-based assay mirroring the in results from the vivoCAIA model for rheumatoid arthritis. Cells were incubated with 10 μM ofthe indicated peptides, as well as 0, 5, 10, or 30 ng/ml IL1β, for 48hours. Upon IL1β stimulation, the cells secrete the metalloproteaseMMP1, which is involved in breaking down the extracellular matrix inarthritis. The assay was done in triplicate and averages with standarddeviations are plotted. SP16 lowered MMP1 secretion compared to thescrambled control peptides, at 20 ng/ml IL1β. (*) indicates p<0.05compared to scrambled peptide control. See, e.g., FIG. 15.

We used the lethal endotoxemia model as a predictor for protectionagainst sepsis during acute radiation syndrome. Lipopolysaccharide (LPS)is an endotoxin purified from gram negative bacteria. In animals, LPSelicits a strong immune response as evidenced by increasedpro-inflammatory serum cytokine levels and lethality at high doses. InAcute Radiation Syndrome, gram negative bacteria leaking from the GIsystem contribute to a systemic inflammatory response syndrome andlethality. In the animal model the peptide was administered at time T=−2hours, T=0 hours or T=0.5 hours. LPS was injected at T=0 hours. Weshowed that SP16 increases survival in lethal endotoxemia therebyallowing us to extrapolate that the peptide would provide treatment inhuman burn victims or in human acute radiation syndrome. Groups of 10animals were injected as indicated, 2 hours before, at the time of,and/or 0.5 hours after induction of lethal endotoxemia. Endotoxemia wasinduced by injection of 15 mg/kg LPS. Survival was monitored at timepoints 4, 8, 24, 28, 32, 48, 52, 56 and 72 hours (FIG. 7).

The NOD model of type I diabetes was used to determine the efficacy ofSP16 in preventing and/or delaying the onset of symptoms and overtdisease. As depicted in FIG. 18, administration of the peptide wasdemonstrated to prevent and delay the development of type I diabetes inthis model.

Therefore, in one aspect we provide methods for treatment of type IIdiabetes, type I diabetes, rheumatoid arthritis and endotoxemia causedby burns and/or radiation comprising administering to a human subject inneed thereof a composition comprising at least one of the peptides ofthe invention. In some aspects, the peptide comprises SP16.

We have shown that the peptides of the invention, e.g., SP16 are tolllike receptor-2 agonists. Accordingly, without wishing to be bound by atheory, the peptides, such as SP16, act as an anti-inflammatory drug bypromoting an anti-inflammatory cytokine profile. Also, without wishingto be bound by a theory, the peptides, such as SP16, also can act asimmune modulators by inducing expansion of tolerogenic and protectiveT-regulatory cells (T-regs). Further, without wishing to be bound by atheory, the peptides, such as SP16, also can down-regulate autoimmuneresponses without inducing general immunological suppression therebyproviding a superior treatment for autoimmune diseases compared to mostof the currently available treatments which generally suppress theimmune system exposing the treated individuals to a risk of infectionswhile treated with the general immunosuppressants.

As the peptides are derived from AAT, and in view of our results in vivoand in vitro models, it is reasonable to expect most of the AAT'stherapeutic effects to apply also to the peptides of the invention, suchas SP16. Specifically, AAT has been shown to modulate T-cellproliferation and NF-kappa-B activation; impair NK target cellinteraction; inhibit serine proteases activation of epithelial cellEGFR/TLR-4 signaling; be involved in TNF-alpha-induced gene expressionand apoptosis or endothelial cells; prevent red blood cell haemolysis byE. coli, decrease circulating eosinophil cell count; inhibit neutrophilchemotaxis, NADHP oxidase and ANCA signaling; inhibit monocyte andmacrophage cytokine release and regulation of CD14 expression, andinhibit mast cell histamine release; and modulate B-cell proliferationand cytokine production.

Therefore, in some embodiments, we provide methods for modulating T-cellproliferation and NF-kappa-B activation; impairing NK target cellinteraction; inhibiting serine proteases activation of epithelial cellEGFR/TLR-4 signaling; modulating TNF-alpha-induced gene expression andapoptosis or endothelial cells; preventing red blood cell haemolysis byE. coli, decreasing circulating eosinophil cell count; inhibitingneutrophil chemotaxis, NADHP oxidase and ANCA signaling; inhibitingmonocyte and macrophage cytokine release and regulation of CD14expression, and inhibiting mast cell histamine release; and modulatingB-cell proliferation and cytokine production comprising the step ofadministering to a human subject a composition comprising at least oneof the peptides of the invention. In some aspects, the peptide is SP16,which may comprise one or more modifications typically performed toenhance peptide bioavailability and/or shelf life, such as pegylationand the like.

We also performed a peptide optimization assay using an alanine scanwith the TLR-2 assay. Data was obtained using an experiment with anengineered TLR-2 indicator cell line (HEK-BLUE™ mTLR2, Invivogen). Cellswere incubated with the 20 μg/ml of the indicated peptides for 24 hours.Upon TLR2 activation, the cells secrete alkaline phosphatase, which canbe assayed. The assay was done in triplicate and averages are plotted.Peptide SP34 is a scrambled peptide control (Yellow), and PAM (Pam3CSK4;Red) is a positive control. See, e.g., FIG. 12.

The following table provides the results from an assay for SP16'spharmacokinetic profile.

PK parameters SP16, IV (5 mg/kg) C0 (μg/m L) 2.5 AUC to Last (μg-hr/mL)0.9 t½ (hr) 1.9 Total CL (mL/hr) 140 Total CL (mL/min/kg) 9.7 Last Timepoint 8.0 MRTINF (hr) 1.1 V (mL) 374 Vss (mL) 149

In performing the assay, three normal rats were injected intravenouslywith 5 mg/kg SP16 and the plasma concentration of SP16 established at 8time points following the injection. For each timepoint, SP16 levelswere determined by LC/MS/MS and the values used to calculate the Cmax(2.5 ug/ml) and T1/2 (1.9 hours). The assay was executed by Apredica,Boston, Mass. Accordingly, we determined that SP16 has a half-life of1.9 hours in normal rats.

The SP16 safety profile also included hERG data. The hERG FastPatchassay showed that SP16 does not inhibit hERG at up to 25 μM doses. Thisdata predicts that SP16 will not have cardiac safety issues in humans.The study was executed by Apredica, Boston, Mass.

Test conc IC50 value Client ID (μM) (μM) comment SP16 0.008-25 >25   Noconcentration-dependent inhibition observed. Quinidine  0.01-10   1.8positive control Customer Mean % Activity Id 0 μM 0.008 μM 0.04 μM 0.2μM 1 μM 5 μM 25 μM Comments SP16 100 89 95 95 78 88 89 Noconcentration-dependent inhibition observed

In addition, we also performed a profile using human receptor palming.The GenSEP Explorer panel contains 111 in vitro assay targets carefullyselected to assess drug/chemical biological activities. Assay categoriesinclude GPCRs, Voltage-Gated Ion Channels, Ligand-Gated Ion Channels,Neurotransmitter transporters, Nuclear Receptors and Steroids as well asa diverse set of biochemical targets including phosphodiesterases,kinases and other relevant enzymes. The study was executed by CaliperLifeSciences, and the results are summarized in the table below. Itappears that SP16 has no effect on 111 human receptors, indicating thatSP16 has an excellent human safety profile.

Number of Percent Receptor Class Receptors Tested inhibitionNeurotransmitter Related 47 Insignificant Steroids 4 Insignificant IonChannels 8 Insignificant Growth factors & hormones 2 InsignificantSecond Messengers 3 Insignificant Brain/gut peptides 15 InsignificantEnzymes 20 Insignificant Enzymes, Kinases 11 Insignificant Cell-Based,Functional 1 Insignificant

In view of the safety profile of SP16, we can reasonably extrapolatethat the other peptide fragments provided herein would also be safe foradministering to human.

In one embodiment, the methods of treatment described herein, furthercomprise selection or diagnosis of a subject having any of theabove-described conditions, e.g., one arising from inflammation prior toadministering a peptide as disclosed herein or a mutant, variant, analogor derivative thereof, to thereby treat the condition or dysfunction,such as inflammation. Such selection is performed by the skilledpractitioner by a number of available methods, for instance, assessmentof symptoms which are described herein. For example, one can assess theamount of TNF-alpha in the subject to determine the amount ofinflammation present in the subject. In the case of diabetes, one canmeasure glycemic control using well-defined blood glucose levels. Insome aspects of all the embodiments of the invention, the peptides canbe administered to individuals with pre-diabetic stage to preventdevelopment of type 2 diabetes. There are a number of tests which can beused to determine if, e.g., a human subject is affected withpre-diabetes. Such tests include, e.g., the A1C test, fasting plasmaglucose test (FPG), and the oral glucose tolerance test (OGTT).Pre-diabetic individuals typically score at or above 5.7% to under 6.5%on the A1C test, whereas diabetics score over 6.5% on this test.Pre-diabetic individuals also typically score at or over 100 mg/dl tounder 126 mg/dl using the FPG test whereas diabetics score over 126mg/dl. Pre-diabetic individuals typically score at or over 140 to under200 mg/dl using the OGTT test whereas diabetic individuals score over200 mg/dl. Thus, a method of preventing diabetes according to thepresent invention may comprise administering the peptide to anindividual who has first been diagnosed as pre-diabetic, e.g., using anyof the above-described methods. In some aspects, the method comprisesidentifying diabetes type 2 in the subject and then administering thesynthetic peptide of the invention to the subject.

In some aspects of all the embodiments, one may use C-reactive proteinas a marker for inflammation or treatment efficacy. C-reactive protein(CRP) is used to detect inflammation if there is a high suspicion oftissue injury or infection somewhere in the body. CRP serves as ageneral marker for infection and inflammation and can be used toevaluate an individual for an acute or chronic inflammatory condition. Ahigh or increasing amount of CRP in the blood suggests the presence ofinflammation. In individuals suspected of having a serious bacterialinfection, a high CRP suggests the presence of one. In people withchronic inflammatory conditions, high levels of CRP suggest a flare-upor that treatment has not been effective. Normal concentration inhealthy human serum is usually lower than 10 mg/L, slightly increasingwith aging. Higher levels are found in late pregnant women, mildinflammation and viral infections (10-40 mg/L), active inflammation,bacterial infection (40-200 mg/L), severe bacterial infections and burns(>200 mg/L). In some aspects the term “reference value” refers to themeasurements of CRP when CRP is used as a diagnostic test forinflammation.

In some aspects, the subject may carry a diagnosed gene mutation, suchas when the subject is diagnosed as having cystic fibrosis (CF). CF isan autosomal, recessive disease caused by mutations in the gene for theprotein cystic fibrosis transmembrane conductance regulator (CFTR). Insome aspects, the invention provides a method of first identifying a CFcausing mutation in the subject and then administering the syntheticpeptide of the invention into the subject. Currently 1913 mutations havebeen reported to the publicly available CF database maintained by CysticFibrosis Centre at the Hospital for Sick Children in Toronto, Canada.Often, testing is used to analyze the most common mutations such asΔF508. The parental history and ethnic origin may provide clues to thekinds of mutations one can screen for in an affected child. Diagnosis ofCF can also include detection of salty tasting skin, poor growth andpoor weight gain despite a normal food intake, accumulation of thick,sticky mucus, frequent chest infections, and coughing or shortness ofbreath. Males can be infertile due to congenital absence of the vasdeferens. Symptoms often appear in infancy and childhood, such as bowelobstruction due to meconium ileus in newborn babies. As the childrengrow, they must exercise to release mucus in the alveoli. Ciliatedepithelial cells in the patient have a mutated protein that leads toabnormally viscous mucus production

In some aspects, the subject is diagnosed with burns and thus at highrisk of developing burn-related endotoxemia, and subsequently, thepeptides of the invention may be administered to the subject to preventdevelopment of endotoxemia. In some aspects, the burn victim does nothave and has not been treated for inflammatory conditions, diabetes,COPD or CF. In some aspects, the subject has been exposed to radiationand has acute radiation injury, and subsequently, the peptides of thepresent invention may be administered to the subject to preventdevelopment of endotoxemia. In some aspects, the subject has signs orsymptoms of endotoxemia and the peptides of the invention may beadministered to such a subject as well. After ionizing radiationexposure, if the dose is sufficiently high, gram negative bacteria leakfrom the gastro-intestinal system and can cause endotoxemia (Sepsis). Wehave shown that the peptides improve survival during induced lethalendotoxemia in a mouse model. Thus, based on these results we canconclude that the peptides can also be used in humans being at risk of asimilar condition, namely endotoxemia or sepsis as a result of burns oracute exposure to radiation.

Successful or effective treatment is evidenced by amelioration of one ormore symptoms of the condition or dysfunction as discussed herein.Administering a peptide as disclosed herein or a mutant, variant, analogor derivative thereof in a subject in need thereof is expected toprevent or retard the development of the conditions and physicaldysfunctions described herein (e.g., those arising from inflammation orauto-immune tissue destruction or to a condition ameliorated bystimulation of expansion of beta cell mass in an individual withdiabetes). The term “prevention” is used to refer to a situation whereina subject does not yet have the specific condition being prevented,meaning that it has not manifested in any appreciable form. Preventionencompasses prevention or slowing of onset and/or severity of a symptom,(including where the subject already has one or more symptoms of anothercondition). Prevention is performed generally in a subject who is atrisk for development of a condition or physical dysfunction. Suchsubjects are said to be in need of prevention. For example, reduction inthe TNF-alpha levels compared to the levels prior to administering thepeptides of the invention, would be evidence of successful treatment.Improvement in glycemic control is another way of showing that thetreatment has had an effect. Also, if the A1C test, fasting plasmaglucose test (FPG), and the oral glucose tolerance test (OGTT) show thatthe pre-diabetic test results stay at pre-diabetic test levels, one canconclude that prevention of diabetes in the pre-diabetic subject hasbeen successful. Also, if a burn victim or a subject exposed to acuteradiation injury does not develop endotoxemia or the signs and symptomsof endotoxemia are mild the administration of the peptide can beconsidered as having an effect in prevention.

In one embodiment, the methods of prevention described herein, furthercomprise selection of such a subject at risk for a condition, e.g.,those arising from inflammation or auto-immune tissue destruction or acondition ameliorated by stimulation of expansion of beta cell mass inan individual with diabetes, or subject that has severe burns or hassuffered acute radiation injury and is thus susceptible for endotoxemia,or a subject that is a smoker and thus susceptible for COPD, or physicaldysfunction as described herein, prior to administering a peptide or amutant, variant, analog or derivative thereof, in the subject, tothereby prevent the condition or dysfunction. Such selection isperformed by the skilled practitioner by a number of available methods.For instance, assessment of risk factors or diagnosis of a disease whichis known to cause the condition or dysfunction, or treatment or therapyknown to cause the condition or dysfunction. Subjects which have adisease or injury or a relevant family history which is known tocontribute to the condition are generally considered to be at increasedrisk.

As used herein, the terms “treat” or “treatment” or “treating” refers totherapeutic treatment measures, wherein the object is to prevent or slowthe development of the disease, such as reducing at least one effect orsymptom of a condition, disease or disorder associated withinflammation. Treatment is generally “effective” if one or more symptomsare improved or clinical markers, such as TNF-alpha, CRP, blood glucoseand/or HbA1c, levels are within normal values or closer to the normalreference values than abnormal values reflecting inflammation or poorglycemic control, depending on the condition, as that term is definedherein. Alternatively, treatment is “effective” if the progression of adisease is slowed down, exhibition of a symptom or a marker for adisease is reduced. That is, “treatment” includes the improvement ofsymptoms or markers, slowing of progress or slowing of worsening of atleast one symptom that would be expected in absence of treatment.Beneficial or desired clinical results include, but are not limited to,alleviation of one or more symptom(s), diminishment of extent ofdisease, stabilized (i.e., not worsening) state of disease, delay orslowing of disease progression, amelioration or palliation of thedisease state. “Treatment” can also mean prolonging survival as comparedto expected survival if not receiving treatment. Those in need oftreatment include patients with one or more symptom of inflammation,such as symptoms associated with rheumatoid arthritis, COPD or diabetes,including Type 1 and Type 2 diabetes. In some aspects of all theembodiments of the invention, the subject in need of treatment hascystic fibrosis or has been subject to burns or acute radiation and isthus at high risk of developing endotoxemia.

TNF-alpha levels can be assessed, for example, using any number ofreadily available commercial ELISA kits. A1C test, FPG, and OGTT arecommonly used to assess glycemic control in diagnosing and managingdiabetes and pre-diabetes.

In some aspects, the invention relates to methods of preventinginflammation by administering the peptides as described to an individualnot yet presenting symptoms of inflammation. For example, the peptidescan be administered to an individual at high risk of developing diabetesor diagnosed with pre-diabetes, a condition defined by increase bloodsugar levels but levels that are not yet considered diabetic, but notyet having diabetes to assist in slowing down the development orpreventing the development of diabetes from the pre-diabetic stage.

Before people develop type 2 diabetes, they almost always have“prediabetes”—blood glucose levels that are higher than normal but notyet high enough to be diagnosed as diabetes. Recent research has shownthat some long-term damage to the body, especially the heart andcirculatory system, may already be occurring during prediabetes. Thereare a number of tests which can be used to determine if, e.g., a humansubject is affected with prediabetes. Such tests include, e.g., the A1Ctest, fasting plasma glucose test (FPG), and the oral glucose tolerancetest (OGTT). Prediabetic individuals typically score at or above 5.7% tounder 6.5% on the A1C test, whereas diabetics score over 6.5% on thistest. Prediabetic individuals also typically score at or over 100 mg/dlto under 126 mg/dl using the FPG test whereas diabetics score over 126mg/dl. Prediabetic individuals typically score at or over 140 to under200 mg/dl using the OGTT test whereas diabetic individuals score over200 mg/dl. Thus, a method of preventing diabetes according to thepresent invention may comprise administering the peptide to anindividual who has been diagnosed as pre-diabetic, e.g., using any ofthe above-described methods.

The term “effective amount” as used herein refers to the amount of apharmaceutical composition comprising one or more peptides as disclosedherein or a mutant, variant, analog or derivative thereof, to decreaseat least one or more symptom of the disease or disorder, and relates toa sufficient amount of pharmacological composition to provide thedesired effect. The phrase “therapeutically effective amount” as usedherein means a sufficient amount of the composition to treat a disorder,at a reasonable benefit/risk ratio applicable to any medical treatment.The term “therapeutically effective amount” therefore refers to anamount of the composition as disclosed herein that is sufficient toeffect a therapeutically or prophylactically reduction in a symptom orclinical marker associated with increased levels of inflammation orhyperglycemia when administered to a typical subject who has anemia,anemia of inflammation or type I diabetes. Typically reduction of morethan 20% of a disease marker, such as an inflammatory marker, e.g.,TNF-alpha, is indicative of effective treatment. In some instances,reduction of more than 50% or more than 75% from the amount of TNF-alphalevels in the individual prior to administering the peptides of theinvention is indicative of effective treatment.

A therapeutically or prophylactically significant reduction in a symptomis, e.g. at least about 10%, at least about 20%, at least about 30%, atleast about 40%, at least about 50%, at least about 60%, at least about70%, at least about 80%, at least about 90%, at least about 100%, atleast about 125%, at least about 150% or more in a measured parameter ascompared to a control or non-treated subject or the state of the subjectprior to administering the peptide. Measured or measurable parametersinclude clinically detectable markers of disease, for example, elevatedor depressed levels of a biological marker, such as TNF-alpha, as wellas parameters related to a clinically accepted scale of symptoms ormarkers for inflammation. It will be understood, however, that the totaldaily usage of the compositions and formulations as disclosed hereinwill be decided by the attending physician within the scope of soundmedical judgment. The exact amount required will vary depending onfactors such as the type of disease being treated, gender, age, andweight of the subject.

With reference to the treatment of a subject with inflammation,auto-immune tissue destruction or type I diabetes, the term“therapeutically effective amount” refers to the amount that is safe andsufficient to delay the development of one or more symptom and resultsin decrease in the amount of an inflammatory marker, e.g., TNF-α or CRPconcentrations, or improvement in blood glucose levels in patientscompared to the amount of the inflammatory marker or blood glucoselevels prior to administering the peptide. The amount can thus improveor cause a decrease in at least one symptom of inflammation, auto-immunetissue destruction or type I diabetes or slow the course of diseaseprogression, such as stabilizing blood glucose levels. The effectiveamount for the treatment of a disease depends on the type of disease,the species being treated, the age and general condition of the subject,the mode of administration and so forth. Thus, it is not possible tospecify the exact “effective amount.” However, for any given case, anappropriate “effective amount” can be determined by one of ordinaryskill in the art using only routine experimentation. The efficacy oftreatment can be judged by an ordinarily skilled practitioner, forexample, efficacy can be assessed in known animal models of inflammation(e.g. LPS model), auto-immune tissue destruction (e.g. CAIA model) ordiabetes (e.g. db/db mouse model). When using an experimental animalmodel, efficacy of treatment is evidenced when a reduction in a symptomof inflammation, auto-immune tissue destruction or hyperglycemia isshown versus untreated animals.

As used herein, the terms “administering,” and “introducing” are usedinterchangeably herein and refer to the placement of the therapeuticagents such as one or more peptides as disclosed herein or a mutant,variant, analog or derivative thereof into a subject by a method orroute which results in delivering of such agent(s) at a desired site.The compounds can be administered by any appropriate route which resultsin an effective treatment in the subject.

The one or more peptides as disclosed herein or a mutant, variant,analog or derivative thereof may be administered by any route known inthe art or described herein, for example, oral, parenteral (e.g.,intravenously or intramuscularly), intraperitoneal, rectal, cutaneous,nasal, vaginal, inhalant, skin (patch), or ocular. The one or morepeptides as disclosed herein or a mutant, variant, analog or derivativethereof may be administered in any dose or dosing regimen. One can alsouse pumps, like the ones used for insulin administration. In someembodiments, the one or more peptides can be administered orally. Asdepicted in FIG. 16, oral administration of SP16 was demonstrated to beefficacious, e.g. compared to intraperitoneal injection of SP16 anddexamethasone.

Dosage

With respect to the therapeutic methods of the invention, it is notintended that the administration of the one or more peptides asdisclosed herein or a mutant, variant, analog or derivative thereof andbe limited to a particular mode of administration, dosage, or frequencyof dosing; the present invention contemplates all modes ofadministration, including intramuscular, intravenous, intraperitoneal,intravesicular, intraarticular, intralesional, subcutaneous, or anyother route sufficient to provide a dose adequate to treat theinflammation-related disorder. The therapeutic may be administered tothe patient in a single dose or in multiple doses. When multiple dosesare administered, the doses may be separated from one another by, forexample, one hour, three hours, six hours, eight hours, one day, twodays, one week, two weeks, or one month. For example, the therapeuticmay be administered for, e.g., 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, or moreweeks. It is to be understood that, for any particular subject, specificdosage regimes should be adjusted over time according to the individualneed and the professional judgment of the person administering orsupervising the administration of the compositions. For example, thedosage of the therapeutic can be increased if the lower dose does notprovide sufficient therapeutic activity.

While the attending physician ultimately will decide the appropriateamount and dosage regimen, therapeutically effective amounts of the oneor more peptides as disclosed herein or a mutant, variant, analog orderivative thereof may be provided at a dose of 0.0001, 0.01, 0.01 0.1,1, 5, 10, 25, 50, 100, 500, or 1,000 mg/kg or μg/kg. Effective doses maybe extrapolated from dose-response curves derived from in vitro oranimal model test bioassays or systems.

Dosages for a particular patient or subject can be determined by one ofordinary skill in the art using conventional considerations, (e.g. bymeans of an appropriate, conventional pharmacological protocol). Aphysician may, for example, prescribe a relatively low dose at first,subsequently increasing the dose until an appropriate response isobtained. The dose administered to a patient is sufficient to effect abeneficial therapeutic response in the patient over time, or, e.g., toreduce symptoms, or other appropriate activity, depending on theapplication. The dose is determined by the efficacy of the particularformulation, and the activity, stability or serum half-life of the oneor more peptides as disclosed herein or a mutant, variant, analog orderivative thereof and the condition of the patient, as well as the bodyweight or surface area of the patient to be treated. The size of thedose is also determined by the existence, nature, and extent of anyadverse side-effects that accompany the administration of a particularvector, formulation, or the like in a particular subject. Therapeuticcompositions comprising one or more peptides as disclosed herein or amutant, variant, analog or derivative thereof are optionally tested inone or more appropriate in vitro and/or in vivo animal models ofdisease, such as models of inflammation or diabetes, to confirmefficacy, tissue metabolism, and to estimate dosages, according tomethods well known in the art. In particular, dosages can be initiallydetermined by activity, stability or other suitable measures oftreatment vs. non-treatment (e.g., comparison of treated vs. untreatedcells or animal models), in a relevant assay. Formulations areadministered at a rate determined by the LD50 of the relevantformulation, and/or observation of any side-effects of one or morepeptides as disclosed herein or a mutant, variant, analog or derivativethereof. Administration can be accomplished via single or divided doses.

In determining the effective amount of one or more peptides as disclosedherein or a mutant, variant, analog or derivative thereof to beadministered in the treatment or prophylaxis of disease the physicianevaluates circulating plasma levels, formulation toxicities, andprogression of the disease.

The efficacy and toxicity of the compound can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., ED50 (the dose is effective in 50% of the population) and LD50(the dose is lethal to 50% of the population). The dose ratio of toxicto therapeutic effects is the therapeutic index, and it can be expressedas the ratio, LD50/ED50. Pharmaceutical compositions which exhibit largetherapeutic indices are preferred.

These compounds may be administered to humans and other animals fortherapy by any suitable route of administration that works for smallpeptides, including orally, nasally, as by, for example, a spray,rectally, intravaginally, parenterally, intracisternally and topically,as by powders, ointments or drops, including buccally and sublingually.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular subject, composition, and mode ofadministration, without being toxic to the subject.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compound employed, the age, sex, weight, condition, generalhealth and prior medical history of the patient being treated, and likefactors well known in the medical arts.

Formulation of Pharmaceutical Compositions—“Pharmaceutically AcceptableCarriers”

The administration of one or more peptides as disclosed herein or amutant, variant, analog or derivative thereof may be by any suitablemeans that results in a concentration of the protein that treats thedisorder. The compound may be contained in any appropriate amount in anysuitable carrier substance, and is generally present in an amount of1-95% by weight of the total weight of the composition. The compositionmay be provided in a dosage form that is suitable for the oral,parenteral (e.g., intravenously or intramuscularly), intraperitoneal,rectal, cutaneous, nasal, vaginal, inhalant, skin (patch), or ocularadministration route. Thus, the composition may be in the form of, e.g.,tablets, capsules, pills, powders, granulates, suspensions, emulsions,solutions, gels including hydrogels, pastes, ointments, creams,plasters, drenches, osmotic delivery devices, suppositories, enemas,injectables, implants, sprays, or aerosols. The pharmaceuticalcompositions may be formulated according to conventional pharmaceuticalpractice (see, e.g., Remington: The Science and Practice of Pharmacy,20th edition, 2000, ed. A. R. Gennaro, Lippincott Williams & Wilkins,Philadelphia, and Encyclopedia of Pharmaceutical Technology, eds. J.Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York,incorporated, herein, by reference in its entirety).

Pharmaceutical compositions according to the invention may be formulatedto release the active compound immediately upon administration or at anypredetermined time or time period after administration. The latter typesof compositions are generally known as controlled release formulations,which include (i) formulations that create substantially constantconcentrations of the agent(s) of the invention within the body over anextended period of time; (ii) formulations that after a predeterminedlag time create substantially constant concentrations of the agent(s) ofthe invention within the body over an extended period of time; (iii)formulations that sustain the agent(s) action during a predeterminedtime period by maintaining a relatively constant, effective level of theagent(s) in the body with concomitant minimization of undesirable sideeffects associated with fluctuations in the plasma level of the agent(s)(sawtooth kinetic pattern); (iv) formulations that localize action ofagent(s), e.g., spatial placement of a controlled release compositionadjacent to or in the diseased tissue or organ; (v) formulations thatachieve convenience of dosing, e.g., administering the composition onceper week or once every two weeks; and (vi) formulations that target theaction of the agent(s) by using carriers or chemical derivatives todeliver the therapeutic to a particular target cell type. Administrationof the protein in the form of a controlled release formulation isespecially preferred for compounds having a narrow absorption window inthe gastrointestinal tract or a relatively short biological half-life.

Any of a number of strategies can be pursued in order to obtaincontrolled release in which the rate of release outweighs the rate ofmetabolism of the compound in question. In one example, controlledrelease is obtained by appropriate selection of various formulationparameters and ingredients, including, e.g., various types of controlledrelease compositions and coatings. Thus, the protein is formulated withappropriate excipients into a pharmaceutical composition that, uponadministration, releases the protein in a controlled manner. Examplesinclude single or multiple unit tablet or capsule compositions, oilsolutions, suspensions, emulsions, microcapsules, molecular complexes,microspheres, nanoparticles, patches, and liposomes.

As used herein, the phrases “parenteral administration” and“administered parenterally” as used herein mean modes of administrationother than enteral and topical administration, usually by injection, andincludes, without limitation, intravenous, intramuscular, intraarterial,intrathecal, intraventricular, intracapsular, intraorbital,intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous,subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal,intracerebrospinal, and intrasternal injection and infusion. The phrases“systemic administration,” “administered systemically”, “peripheraladministration” and “administered peripherally” as used herein mean theadministration therapeutic compositions other than directly into a tumorsuch that it enters the animal's system and, thus, is subject tometabolism and other like processes.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio. The phrase“pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in maintaining the activity of or carrying ortransporting the subject agents from one organ, or portion of the body,to another organ, or portion of the body. In addition to being“pharmaceutically acceptable” as that term is defined herein, eachcarrier must also be “acceptable” in the sense of being compatible withthe other ingredients of the formulation. The pharmaceutical formulationcomprising the one or more peptides as disclosed herein or a mutant,variant, analog or derivative thereof in combination with one or morepharmaceutically acceptable ingredients. The carrier can be in the formof a solid, semi-solid or liquid diluent, cream or a capsule. Thesepharmaceutical preparations are a further object of the invention.Usually the amount of active compounds is between 0.1-95% by weight ofthe preparation, preferably between 0.2-20% by weight in preparationsfor parenteral use and preferably between 1 and 50% by weight inpreparations for oral administration. For the clinical use of themethods of the present invention, targeted delivery composition of theinvention is formulated into pharmaceutical compositions orpharmaceutical formulations for parenteral administration, e.g.,intravenous; mucosal, e.g., intranasal; enteral, e.g., oral; topical,e.g., transdermal; ocular, e.g., via corneal scarification or other modeof administration. The pharmaceutical composition contains a compound ofthe invention in combination with one or more pharmaceuticallyacceptable ingredients. The carrier can be in the form of a solid,semi-solid or liquid diluent, cream or a capsule.

The term “pharmaceutically acceptable carriers” is intended to includeall solvents, diluents, or other liquid vehicle, dispersion orsuspension aids, surface active agents, isotonic agents, thickening oremulsifying agents, preservatives, solid binders, lubricants and thelike, as suited to the particular dosage form desired. Typically, suchcompounds are carried or transported from one organ, or portion of thebody, to another organ, or portion of the body. Each carrier must be“acceptable” in the sense of being compatible with the other ingredientsof the formulation and not injurious to the patient. Some examples ofmaterials which can serve as pharmaceutically acceptable carriersinclude: sugars, such as lactose, glucose and sucrose; starches, such ascorn starch and potato starch; cellulose, and its functionalderivatives, such as sodium carboxymethyl cellulose, ethyl cellulose andcellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients,such as cocoa butter and suppository waxes; oils, such as peanut oil,cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; glycols, such as propylene glycol; polyols, such asglycerin, sorbitol, mannitol and polyethylene glycol; esters, such asethyl oleate and ethyl laurate; agar; buffering agents, such asmagnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-freewater; isotonic saline; Ringer's solution; ethyl alcohol; phosphatebuffer solutions; and other non-toxic compatible substances employed inpharmaceutical formulations.

The term “pharmaceutical composition” is used herein refer tocompositions or formulations that usually comprise an excipient, such asa pharmaceutically acceptable carrier that is conventional in the artand that is suitable for administration to mammals, and preferablyhumans or human cells. Such compositions can be specifically formulatedfor administration via one or more of a number of routes, including butnot limited to, oral, ocular, parenteral, intravenous, intraarterial,subcutaneous, intranasal, sublingual, intraspinal,intracerebroventricular, and the like. In addition, compositions fortopical (e.g., oral mucosa, respiratory mucosa) and/or oraladministration can form solutions, suspensions, tablets, pills,capsules, sustained-release formulations, oral rinses, or powders, asknown in the art are described herein. The compositions also can includestabilizers and preservatives. For examples of carriers, stabilizers andadjuvants, University of the Sciences in Philadelphia (2005) Remington:The Science and Practice of Pharmacy with Facts and Comparisons, 21stEd.

In certain embodiments, the compounds of the present invention maycontain one or more acidic functional groups and, thus, are capable offorming pharmaceutically acceptable salts with pharmaceuticallyacceptable bases. The term “pharmaceutically acceptable salts, esters,amides, and prodrugs” as used herein refers to those carboxylate salts,amino acid addition salts, esters, amides, and prodrugs of the compoundsof the present invention which are, within the scope of sound medicaljudgment, suitable for use in contact with the tissues of patientswithout undue toxicity, irritation, allergic response, and the like,commensurate with a reasonable benefit/risk ratio, and effective fortheir intended use of the compounds of the invention. The term “salts”refers to the relatively non-toxic, inorganic and organic acid additionsalts of compounds of the present invention. These salts can be preparedin situ during the final isolation and purification of the compounds orby separately reacting the purified compound in its free base form witha suitable organic or inorganic acid and isolating the salt thus formed.These may include cations based on the alkali and alkaline earth metalssuch as sodium, lithium, potassium, calcium, magnesium and the like, aswell as nontoxic ammonium, quaternary ammonium, and amine cationsincluding, but not limited to ammonium, tetramethylanunonium, tetraethylammonium, methyl amine, dimethyl amine, trimethylamine, triethylamine,ethylamine, and the like (see, e.g., Berge S. M., et al. (1977) J.Pharm. Sci. 66, 1, which is incorporated herein by reference).

The term “pharmaceutically acceptable esters” refers to the relativelynontoxic, esterified products of the compounds of the present invention.These esters can be prepared in situ during the final isolation andpurification of the compounds, or by separately reacting the purifiedcompound in its free acid form or hydroxyl with a suitable esterifyingagent. Carboxylic acids can be converted into esters via treatment withan alcohol in the presence of a catalyst. The term is further intendedto include lower hydrocarbon groups capable of being solvated underphysiological conditions, e.g., alkyl esters, methyl, ethyl and propylesters.

As used herein, “pharmaceutically acceptable salts or prodrugs” aresalts or prodrugs that are, within the scope of sound medical judgment,suitable for use in contact with the tissues of subject without unduetoxicity, irritation, allergic response, and the like, commensurate witha reasonable benefit/risk ratio, and effective for their intended use.

The term “prodrug” refers to compounds that are rapidly transformed invivo to yield the functionally active one or more peptides as disclosedherein or a mutant, variant, analog or derivative thereof. A thoroughdiscussion is provided in T. Higachi and V. Stella, “Pro-drugs as NovelDelivery Systems,” Vol. 14 of the A. C. S. Symposium Series, and inBioreversible Carriers in: Drug Design, ed. Edward B. Roche, AmericanPharmaceutical Association and Pergamon Press, 1987, both of which arehereby incorporated by reference. As used herein, a prodrug is acompound that, upon in vivo administration, is metabolized or otherwiseconverted to the biologically, pharmaceutically or therapeuticallyactive form of the compound. A prodrug of the one or more peptides asdisclosed herein or a mutant, variant, analog or derivative thereof canbe designed to alter the metabolic stability or the transportcharacteristics of one or more peptides as disclosed herein or a mutant,variant, analog or derivative thereof, to mask side effects or toxicity,to improve the flavor of a compound or to alter other characteristics orproperties of a compound. By virtue of knowledge of pharmacodynamicprocesses and drug metabolism in vivo, once a pharmaceutically activeform of the one or more peptides as disclosed herein or a mutant,variant, analog or derivative thereof, those of skill in thepharmaceutical art generally can design prodrugs of the compound (see,e.g., Nogrady (1985) Medicinal Chemistry A Biochemical Approach, OxfordUniversity Press, N.Y., pages 388-392). Conventional procedures for theselection and preparation of suitable prodrugs are described, forexample, in “Design of Prodrugs,” ed. H. Bundgaard, Elsevier, 1985.Suitable examples of prodrugs include methyl, ethyl and glycerol estersof the corresponding acid.

Parenteral Compositions

The pharmaceutical composition may be administered parenterally byinjection, infusion, or implantation (subcutaneous, intravenous,intramuscular, intraperitoneal, or the like) in dosage forms,formulations, or via suitable delivery devices or implants containingconventional, non-toxic pharmaceutically acceptable carriers andadjuvants. The formulation and preparation of such compositions are wellknown to those skilled in the art of pharmaceutical formulation.

Compositions for parenteral use may be provided in unit dosage forms(e.g., in single-dose ampoules), or in vials containing several dosesand in which a suitable preservative may be added (see below). Thecomposition may be in form of a solution, a suspension, an emulsion, aninfusion device, or a delivery device for implantation, or it may bepresented as a dry powder to be reconstituted with water or anothersuitable vehicle before use. Apart from the active agent(s), thecomposition may include suitable parenterally acceptable carriers and/orexcipients. The active agent(s) may be incorporated into microspheres,microcapsules, nanoparticles, liposomes, or the like for controlledrelease. Furthermore, the composition may include suspending,solubilizing, stabilizing, pH-adjusting agents, tonicity adjustingagents, and/or dispersing agents.

As indicated above, the pharmaceutical compositions according to theinvention may be in a form suitable for sterile injection. To preparesuch a composition, the suitable active agent(s) are dissolved orsuspended in a parenterally acceptable liquid vehicle. Among acceptablevehicles and solvents that may be employed are water, water adjusted toa suitable pH by addition of an appropriate amount of hydrochloric acid,sodium hydroxide or a suitable buffer, 1,3-butanediol, Ringer'ssolution, dextrose solution, and isotonic sodium chloride solution. Theaqueous formulation may also contain one or more preservatives (e.g.,methyl, ethyl or n-propyl p-hydroxybenzoate). In cases where one of thecompounds is only sparingly or slightly soluble in water, a dissolutionenhancing or solubilizing agent can be added, or the solvent may include10-60% w/w of propylene glycol or the like.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfate, sodium sulfite and the like;oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations of the present invention include those suitable forintravenous, oral, nasal, topical, transdermal, buccal, sublingual,rectal, vaginal and/or parenteral administration. The formulations mayconveniently be presented in unit dosage form and may be prepared by anymethods well known in the art of pharmacy. The amount of activeingredient which can be combined with a carrier material to produce asingle dosage form will generally be that amount of the compound whichproduces a therapeutic effect.

Methods of preparing these formulations or compositions include the stepof bringing into association a compound of the present invention withthe carrier and, optionally, one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association a compound of the present invention withliquid carriers, or finely divided solid carriers, or both, and then, ifnecessary, shaping the product.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent invention as an active ingredient. A compound of the presentinvention may also be administered as a bolus, electuary or paste.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more peptides as disclosed herein or amutant, variant, analog or derivative thereof in combination with one ormore pharmaceutically acceptable sterile isotonic aqueous or nonaqueoussolutions, dispersions, suspensions or emulsions, or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain antioxidants, buffers,bacteriostats, solutes which render the formulation isotonic with theblood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions comprising one or morepeptides as disclosed herein or a mutant, variant, analog or derivativethereof include water, ethanol, polyols (such as glycerol, propyleneglycol, polyethylene glycol, and the like), and suitable mixturesthereof, vegetable oils, such as olive oil, and injectable organicesters, such as ethyl oleate. Proper fluidity can be maintained, forexample, by the use of coating materials, such as lecithin, by themaintenance of the required particle size in the case of dispersions,and by the use of surfactants.

These compositions can also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsulated matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug, such as one ormore peptides as disclosed herein or a mutant, variant, analog orderivative thereof in liposomes or microemulsions which are compatiblewith body tissue.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms by conventional methodsknown to those of ordinary skill in the art.

Controlled Release Parenteral Compositions

Controlled release parenteral compositions may be in form of aqueoussuspensions, microspheres, microcapsules, magnetic microspheres, oilsolutions, oil suspensions, or emulsions. The composition may also beincorporated in biocompatible carriers, liposomes, nanoparticles,implants, or infusion devices.

Materials for use in the preparation of microspheres and/ormicrocapsules are, e.g., biodegradable/bioerodible polymers such aspolygalactia poly-(isobutyl cyanoacrylate),poly(2-hydroxyethyl-L-glutamnine), poly(lactic acid), polyglycolic acid,and mixtures thereof. Biocompatible carriers that may be used whenformulating a controlled release parenteral formulation arecarbohydrates (e.g., dextrans), proteins (e.g., albumin), lipoproteins,or antibodies. Materials for use in implants can be nonbiodegradable(e.g., polydimethyl siloxane) or biodegradable (e.g.,poly(caprolactone), poly(lactic acid), poly(glycolic acid) or poly(orthoesters)) or combinations thereof.

Solid Dosage Forms for Oral Use

Formulations for oral use include tablets containing the activeingredient(s) in a mixture with non-toxic pharmaceutically acceptableexcipients, and such formulations are known to the skilled artisan(e.g., U.S. Pat. Nos. 5,817,307; 5,824,300; 5,830,456; 5,846,526;5,882,640; 5,910,304; 6,036,949; 6,036,949; and 6,372,218 herebyincorporated by reference). These excipients may be, for example, inertdiluents or fillers (e.g., sucrose, sorbitol, sugar, mannitol,microcrystalline cellulose, starches including potato starch, calciumcarbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate,or sodium phosphate); granulating and disintegrating agents (e.g.,cellulose derivatives including microcrystalline cellulose, starchesincluding potato starch, croscarmellose sodium, alginates, or alginicacid); binding agents (e.g., sucrose, glucose, sorbitol, acacia, alginicacid, sodium alginate, gelatin, starch, pregelatinized starch,microcrystalline cellulose, magnesium aluminum silicate,carboxymethylcellulose sodium, methylcellulose, hydroxypropylmethylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethyleneglycol); and lubricating agents, glidants, and anti-adhesives (e.g.,magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenatedvegetable oils, or talc). Other pharmaceutically acceptable excipientscan be colorants, flavoring agents, plasticizers, humectants, bufferingagents, and the like.

The tablets may be uncoated or they may be coated by known techniques,optionally to delay disintegration and absorption in thegastrointestinal tract and thereby providing a sustained action over alonger period. The coating may be adapted to release the protein in apredetermined pattern (e.g., in order to achieve a controlled releaseformulation) or it may be adapted not to release the agent(s) untilafter passage of the stomach (enteric coating). The coating may be asugar coating, a film coating (e.g., based on hydroxypropylmethylcellulose, methylcellulose, methyl hydroxyethylcellulose,hydroxypropylcellulose, carboxymethylcellulose, acrylate copolymers,polyethylene glycols and/or polyvinylpyrrolidone), or an enteric coating(e.g., based on methacrylic acid copolymer, cellulose acetate phthalate,hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcelluloseacetate succinate, polyvinyl acetate phthalate, shellac, and/orethylcellulose). Furthermore, a time delay material such as, e.g.,glyceryl monostearate or glyceryl distearate, may be employed.

The solid tablet compositions may include a coating adapted to protectthe composition from unwanted chemical changes, (e.g., chemicaldegradation prior to the release of the active substances). The coatingmay be applied on the solid dosage form in a similar manner as thatdescribed in Encyclopedia of Pharmaceutical Technology, supra.

The compositions of the invention may be mixed together in the tablet,or may be partitioned. In one example, a first agent is contained on theinside of the tablet, and a second agent is on the outside, such that asubstantial portion of the second agent is released prior to the releaseof the first agent.

Formulations for oral use may also be presented as chewable tablets, oras hard gelatin capsules wherein the active ingredient is mixed with aninert solid diluent (e.g., potato starch, lactose, microcrystallinecellulose, calcium carbonate, calcium phosphate, or kaolin), or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium, for example, peanut oil, liquid paraffin, or olive oil.Powders and granulates may be prepared using the ingredients mentionedabove under tablets and capsules in a conventional manner using, e.g., amixer, a fluid bed apparatus, or spray drying equipment.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient is mixed with one or more pharmaceutically acceptablecarriers, such as sodium citrate or dicalcium phosphate, and/or any ofthe following: fillers or extenders, such as starches, lactose, sucrose,glucose, mannitol, and/or silicic acid; binders, such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; humectants, such as glycerol; disintegratingagents, such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate; solutionretarding agents, such as paraffin; absorption accelerators, such asquaternary ammonium compounds; wetting agents, such as, for example,cetyl alcohol and glycerol monostearate; absorbents, such as kaolin andbentonite clay; lubricants, such a talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, andmixtures thereof; and coloring agents. In the case of capsules, tabletsand pills, the pharmaceutical compositions may also comprise bufferingagents. Solid compositions of a similar type may also be employed asfillers in soft and hard-filled gelatin capsules using such excipientsas lactose or milk sugars, as well as high molecular weight polyethyleneglycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients. Inone aspect, a solution of resolvin and/or protectin or precursor oranalog thereof can be administered as eye drops for ocularneovascularization or ear drops to treat otitis.

Oral administration of peptides has been shown to work for other proteinor peptide drugs as well. For example, oral administration of ananti-CD3 antibody has been shown to work in treatment of, for example,diabetes (Ishikawa et al. Diabetes. 2007 August; 56(8):2103-9. Epub 2007Apr. 24), and autoimmune encephalomyelitis (Ochi et al. Nat Med. 2006June; 12(6):627-35. Epub 2006 May 21). Without wishing to be bound by atheory, we suggest that this is possible via the gut associated lymphoidtissue (GALT). Accordingly, in some aspects of all the embodiments ofthe invention, the formulation of the peptides is oral formulation, andthe methods are performed by administering the peptides orally.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs.

In addition to the active ingredient, the liquid dosage forms maycontain inert diluents commonly used in the art, such as, for example,water or other solvents, solubilizing agents and emulsifiers, such asethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils(in particular, cottonseed, groundnut, corn, germ, olive, castor andsesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. Besides inertdiluents, the oral compositions can also include adjuvants such aswetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Dosage forms for the topical or transdermal administration of one ormore peptides as disclosed herein or a mutant, variant, analog orderivative thereof include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active compound maybe mixed under sterile conditions with a pharmaceutically acceptablecarrier, and with any preservatives, buffers, or propellants, which maybe required.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof. Powders and sprays cancontain, in addition to a compound of this invention, excipients such aslactose, talc, silicic acid, aluminum hydroxide, calcium silicates andpolyamide powder, or mixtures of these substances. Sprays canadditionally contain customary propellants, such aschlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, suchas butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of the compounds (resolvins and/or protectins and/or precursorsor analogues thereof) of the present invention to the body. Such dosageforms can be made by dissolving or dispersing the compound in the propermedium. Absorption enhancers can also be used to increase the flux ofthe compound across the skin. The rate of such flux can be controlled byeither providing a rate controlling membrane or dispersing the activecompound in a polymer matrix or gel. In another aspect, biodegradable orabsorbable polymers can provide extended, often localized, release ofpolypeptide agents. The potential benefits of an increased half-life orextended release for a therapeutic agent are clear. A potential benefitof localized release is the ability to achieve much higher localizeddosages or concentrations, for greater lengths of time, relative tobroader systemic administration, with the potential to also avoidpossible undesirable side effects that may occur with systemicadministration.

Bioabsorbable polymeric matrix suitable for delivery of the one or morepeptides as disclosed herein or a mutant, variant, analog or derivativethereof can be selected from a variety of synthetic bioabsorbablepolymers, which are described extensively in the literature. Suchsynthetic bioabsorbable, biocompatible polymers, which may releaseproteins over several weeks or months can include, for example,poly-a-hydroxy acids (e.g. polylactides, polyglycolides and theircopolymers), polyanhydrides, polyorthoesters, segmented block copolymersof polyethylene glycol and polybutylene terephthalate (Polyactive™),tyrosine derivative polymers or poly(ester-amides). Suitablebioabsorbable polymers to be used in manufacturing of drug deliverymaterials and implants are discussed e.g. in U.S. Pat. Nos. 4,968,317,5,618,563, among others, and in “Biomedical Polymers” edited by S. W.Shalaby, Carl Hanser Verlag, Munich, Vienna, New York, 1994 and in manyreferences cited in the above publications. The particular bioabsorbablepolymer that should be selected will depend upon the particular patientthat is being treated.

Gene Therapy

One or more peptides as disclosed herein or a mutant, variant, analog orderivative thereof can be effectively used in treatment by gene therapy.See, generally, for example, U.S. Pat. No. 5,399,346, which isincorporated herein by reference. The general principle is to introducethe polynucleotide into a target cell in a patient.

Entry into the cell is facilitated by suitable techniques known in theart such as providing the polynucleotide in the form of a suitablevector, or encapsulation of the polynucleotide in a liposome.

A desired mode of gene therapy is to provide the polynucleotide in sucha way that it will replicate inside the cell, enhancing and prolongingthe desired effect. Thus, the polynucleotide is operably linked to asuitable promoter, such as the natural promoter of the correspondinggene, a heterologous promoter that is intrinsically active in liver,neuronal, bone, muscle, skin, joint, or cartilage cells, or aheterologous promoter that can be induced by a suitable agent.

Expression vectors compatible with eukaryotic cells, preferably thosecompatible with vertebrate cells, can be used to produce recombinantconstructs for the expression of one or more peptides as disclosedherein or a mutant, variant, analog or derivative thereof, includingfusion proteins with one or more peptides as disclosed herein or amutant, variant, analog or derivative thereof. Eukaryotic cellexpression vectors are well known in the art and are available fromseveral commercial sources. Typically, such vectors are providedcontaining convenient restriction sites for insertion of the desired DNAsegment. These vectors can be viral vectors such as adenovirus,adeno-associated virus, pox virus such as an orthopox (vaccinia andattenuated vaccinia), avipox, lentivirus, murine moloney leukemia virus,etc. Alternatively, plasmid expression vectors can be used.

Viral vector systems which can be utilized in the present inventioninclude, but are not limited to, (a) adenovirus vectors; (b) retrovirusvectors; (c) adeno-associated virus vectors; (d) herpes simplex virusvectors; (e) SV 40 vectors; (f) polyoma virus vectors; (g) papillomavirus vectors; (h) picornavirus vectors; (i) pox virus vectors such asan orthopox, e.g., vaccinia virus vectors or avipox, e.g. canary pox orfowl pox; and (j) a helper-dependent or gutless adenovirus. In apreferred embodiment, the vector is an adenovirus. Replication-defectiveviruses can also be advantageous.

The vector may or may not be incorporated into the cells genome. Theconstructs may include viral sequences for transfection, if desired.Alternatively, the construct may be incorporated into vectors capable ofepisomal replication, e.g. EPV and EBV vectors.

By “operably linked” is meant that a nucleic acid molecule and one ormore regulatory sequences (e.g., a promoter) are connected in such a wayas to permit expression and/or secretion of the product (e.g., aprotein) of the nucleic acid molecule when the appropriate molecules(e.g., transcriptional activator proteins) are bound to the regulatorysequences. Stated another way, the term “operatively linked” as usedherein refers to the functional relationship of the nucleic acidsequences with regulatory sequences of nucleotides, such as promoters,enhancers, transcriptional and translational stop sites, and othersignal sequences. For example, operative linkage of nucleic acidsequences, typically DNA, to a regulatory sequence or promoter regionrefers to the physical and functional relationship between the DNA andthe regulatory sequence or promoter such that the transcription of suchDNA is initiated from the regulatory sequence or promoter, by an RNApolymerase that specifically recognizes, binds and transcribes the DNA.In order to optimize expression and/or in vitro transcription, it may benecessary to modify the regulatory sequence for the expression of thenucleic acid or DNA in the cell type for which it is expressed. Thedesirability of, or need of, such modification may be empiricallydetermined. An operatively linked polynucleotide which is to beexpressed typically includes an appropriate start signal (e.g., ATG) andmaintains the correct reading frame to permit expression of thepolynucleotide sequence under the control of the expression controlsequence, and production of the desired polypeptide encoded by thepolynucleotide sequence.

As used herein, the terms “promoter” or “promoter region” or “promoterelement” have been defined herein, refers to a segment of a nucleic acidsequence, typically but not limited to DNA or RNA or analogues thereof,that controls the transcription of the nucleic acid sequence to which itis operatively linked. The promoter region includes specific sequencesthat are sufficient for RNA polymerase recognition, binding andtranscription initiation. This portion of the promoter region isreferred to as the promoter. In addition, the promoter region includessequences which modulate this recognition, binding and transcriptioninitiation activity of RNA polymerase. These sequences may be ds-actingor may be responsive to trans-acting factors. Promoters, depending uponthe nature of the regulation may be constitutive or regulated.

The term “regulatory sequences” is used interchangeably with “regulatoryelements” herein refers element to a segment of nucleic acid, typicallybut not limited to DNA or RNA or analogues thereof, that modulates thetranscription of the nucleic acid sequence to which it is operativelylinked, and thus act as transcriptional modulators. Regulatory sequencesmodulate the expression of gene and/or nucleic acid sequence to whichthey are operatively linked. Regulatory sequence often comprise“regulatory elements” which are nucleic acid sequences that aretranscription binding domains and are recognized by the nucleicacid-binding domains of transcriptional proteins and/or transcriptionfactors, repressors or enhancers etc. Typical regulatory sequencesinclude, but are not limited to, transcriptional promoters, induciblepromoters and transcriptional elements, an optional operate sequence tocontrol transcription, a sequence encoding suitable mRNA ribosomalbinding sites, and sequences to control the termination of transcriptionand/or translation. Included in the term “regulatory elements” arenucleic acid sequences such as initiation signals, enhancers, andpromoters, which induce or control transcription of protein codingsequences with which they are operatively linked. In some examples,transcription of a recombinant gene is under the control of a promotersequence (or other transcriptional regulatory sequence) which controlsthe expression of the recombinant gene in a cell-type in whichexpression is intended. It will also be understood that the recombinantgene can be under the control of transcriptional regulatory sequenceswhich are the same or which are different from those sequences whichcontrol transcription of the naturally-occurring form of a protein. Insome instances the promoter sequence is recognized by the syntheticmachinery of the cell, or introduced synthetic machinery, required forinitiating transcription of a specific gene.

Regulatory sequences can be a single regulatory sequence or multipleregulatory sequences, or modified regulatory sequences or fragmentsthereof. Modified regulatory sequences are regulatory sequences wherethe nucleic acid sequence has been changed or modified by some means,for example, but not limited to, mutation, methylation etc.

Regulatory sequences useful in the methods as disclosed herein arepromoter elements which are sufficient to render promoter-dependent geneexpression controllable for cell type-specific, tissue-specific orinducible by external signals or agents (e g enhancers or repressors);such elements may be located in the 5′ or 3′ regions of the native gene,or within an intron.

As used herein, the term “tissue-specific promoter” means a nucleic acidsequence that serves as a promoter, i.e., regulates expression of aselected nucleic acid sequence operably linked to the promoter, andwhich selectively affects expression of the selected nucleic acidsequence in specific cells of a tissue.

In some embodiments, it can be advantageous to direct expression of oneor more peptides as disclosed herein or a mutant, variant, analog orderivative thereof in a tissue- or cell-specific manner. Muscle-specificexpression can be achieved, for example, using the skeletal muscle MKCpromoter (as disclosed in U.S. Patent Application WO2007/100722, whichis incorporated herein by reference), or other muscle-specificpromoters, such as α-myosin heavy chain, myosin light chain-2 (which isspecific for skeletal muscle (Shani et al., Nature, 314;283-86, 1985),gonadotrophic releasing hormone gene control region which is active inthe hypothalamus (Mason et al, Science, 234;1372-78, 1986), and smoothmuscle promoter SM22a, which are all commonly known in the art.

The term “constitutively active promoter” refers to a promoter of a genewhich is expressed at all times within a given cell. Exemplary promotersfor use in mammalian cells include cytomegalovirus (CMV), and for use inprokaryotic cells include the bacteriophage T7 and T3 promoters, and thelike. The term “inducible promoter” refers to a promoter of a gene whichcan be expressed in response to a given signal, for example addition orreduction of an agent. Non-limiting examples of an inducible promoterare “tet-on” and “tet-off’ promoters, or promoters that are regulated ina specific tissue type.

In a specific embodiment, viral vectors that contain nucleic acidsequences encoding the one or more peptides as disclosed herein or amutant, variant, analog or derivative thereof are used. For example, aretroviral vector can be used (see Miller et al., Meth. Enzymol.217:581-599 (1993)). These retroviral vectors contain the componentsnecessary for the correct packaging of the viral genome and integrationinto the host cell DNA. More detail about retroviral vectors can befound in Boesen et al., Biotherapy 6:291-302 (1994), which describes theuse of a retroviral vector to deliver the mdrl gene to hematopoieticstem cells in order to make the stem cells more resistant tochemotherapy. Other references illustrating the use of retroviralvectors in gene therapy are: Clowes et al., J. Clin. Invest. 93:644-651(1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg,Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr.Opin. in Genetics and Devel. 3:110-114 (1993).

The production of a recombinant retroviral vector carrying a gene ofinterest is typically achieved in two stages. First, sequence encodingone or more peptides as disclosed herein or a mutant, variant, analog orderivative thereof can be inserted into a retroviral vector whichcontains the sequences necessary for the efficient expression of themetabolic regulators (including promoter and/or enhancer elements whichcan be provided by the viral long terminal repeats (LTRs) or by aninternal promoter/enhancer and relevant splicing signals), sequencesrequired for the efficient packaging of the viral RNA into infectiousvirions (e.g., a packaging signal (Psi), a tRNA primer binding site(−PBS), a 3′ regulatory sequence required for reverse transcription(+PBS)), and a viral LTRs). The LTRs contain sequences required for theassociation of viral genomic RNA, reverse transcriptase and integrasefunctions, and sequences involved in directing the expression of thegenomic RNA to be packaged in viral particles.

Following the construction of the recombinant retroviral vector, thevector DNA is introduced into a packaging cell line. Packaging celllines provide viral proteins required in trans for the packaging ofviral genomic RNA into viral particles having the desired host range(e.g., the viral-encoded core (gag), polymerase (pol) and envelope (env)proteins). The host range is controlled, in part, by the type ofenvelope gene product expressed on the surface of the viral particle.Packaging cell lines can express ecotrophic, amphotropic or xenotropicenvelope gene products. Alternatively, the packaging cell line can lacksequences encoding a viral envelope (env) protein. In this case, thepackaging cell line can package the viral genome into particles whichlack a membrane-associated protein (e.g., an env protein). To produceviral particles containing a membrane-associated protein which permitsentry of the virus into a cell, the packaging cell line containing theretroviral sequences can be transfected with sequences encoding amembrane-associated protein (e.g., the G protein of vesicular stomatitisvirus (VSV)). The transfected packaging cell can then produce viralparticles which contain the membrane-associated protein expressed by thetransfected packaging cell line; these viral particles which containviral genomic RNA derived from one virus encapsidated by the envelopeproteins of another virus are said to be pseudotyped virus particles.

Adenoviruses are other viral vectors that can be used in gene therapy.Adenoviruses are especially attractive vehicles for delivering genes torespiratory epithelia. Adenoviruses naturally infect respiratoryepithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, CurrentOpinion in Genetics and Development 3:499-503 (1993) present a review ofadenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10(1994) demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Another preferred viralvector is a pox virus such as a vaccinia virus, for example anattenuated vaccinia such as Modified Virus Ankara (MVA) or NYVAC, anavipox such as fowl pox or canary pox. Other instances of the use ofadenoviruses in gene therapy can be found in Rosenfeld et al., Science252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992);Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT PublicationWO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). In anotherembodiment, lentiviral vectors are used, such as the HIV based vectorsdescribed in U.S. Pat. Nos. 6,143,520; 5,665,557; and 5,981,276, whichare herein incorporated by reference. Use of Adeno-associated virus(AAV) vectors is also contemplated (Walsh et al., Proc. Soc. Exp. Biol.Med. 204:289-300 (1993); and U.S. Pat. No. 5,436,146, which areincorporated herein by reference).

Another approach to gene therapy involves transferring a gene to cellsin tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a patient.

U.S. Pat. No. 5,676,954 (which is herein incorporated by reference)reports on the injection of genetic material, complexed with cationicliposome carriers, into mice. U.S. Pat. Nos. 4,897,355, 4,946,787,5,049,386, 5,459,127, 5,589,466, 5,693,622, 5,580,859, 5,703,055, andinternational publication NO: WO 94/9469 (which are herein incorporatedby reference) provide cationic lipids for use in transfecting DNA intocells and mammals. U.S. Pat. Nos. 5,589,466, 5,693,622, 5,580,859,5,703,055, and international publication NO: WO 94/9469 (which areherein incorporated by reference) provides methods for deliveringDNA-cationic lipid complexes to mammals. Such cationic lipid complexesor nanoparticles can also be used to deliver protein.

A gene or nucleic acid sequence can be introduced into a target cell byany suitable method. For example, one or more peptides as disclosedherein or a mutant, variant, analog or derivative thereof constructs canbe introduced into a cell by transfection (e.g., calcium phosphate orDEAE-dextran mediated transfection), lipofection, electroporation,microinjection (e.g., by direct injection of naked DNA), biolistics,infection with a viral vector containing a muscle related transgene,cell fusion, chromosome-mediated gene transfer, microcell-mediated genetransfer, nuclear transfer, and the like. A nucleic acid encoding one ormore peptides as disclosed herein or a mutant, variant, analog orderivative thereof can be introduced into cells by electroporation (see,e.g., Wong and Neumann, Biochem. Biophys. Res. Commun. 107:584-87(1982)) and biolistics (e.g., a gene gun; Johnston and Tang, MethodsCell Biol. 43 Pt A:353-65 (1994); Fynan et al., Proc. Natl. Acad. Sci.USA 90:11478-82 (1993)).

In certain embodiments, a gene or nucleic acid sequence encoding one ormore peptides as disclosed herein or a mutant, variant, analog orderivative thereof can be introduced into target cells by transfectionor lipofection. Suitable agents for transfection or lipofection include,for example, calcium phosphate, DEAE dextran, lipofectin, lipfectamine,DIMRIE C, Superfect, and Effectin (Qiagen), unifectin, maxifectin,DOTMA, DOGS (Transfectam; dioctadecylamidoglycylspermine), DOPE(1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DOTAP(1,2-dioleoyl-3-trimethylammonium propane), DDAB (dimethyldioctadecylammonium bromide), DHDEAB(N,N-di-n-hexadecyl-N,N-dihydroxyethyl ammonium bromide), HDEAB(N-n-hexadecyl-N,N-dihydroxyethylammonium bromide), polybrene,poly(ethylenimine) (PEI), and the like. (See, e.g., Banerjee et al.,Med. Chem. 42:4292-99 (1999); Godbey et al., Gene Ther. 6:1380-88(1999); Kichler et al., Gene Ther. 5:855-60 (1998); Birchaa et al., J.Pharm. 183:195-207 (1999), incorporated herein by reference in theirentireties).

Methods known in the art for the therapeutic delivery of agents such asproteins and/or nucleic acids can be used for the delivery of apolypeptide or nucleic acid encoding one or more peptides as disclosedherein or a mutant, variant, analog or derivative thereof, e.g.,cellular transfection, gene therapy, direct administration with adelivery vehicle or pharmaceutically acceptable carrier, indirectdelivery by providing recombinant cells comprising a nucleic acidencoding a targeting fusion polypeptide of the invention.

Various delivery systems are known and can be used to directlyadminister therapeutic polypeptides such as the one or more peptides asdisclosed herein or a mutant, variant, analog or derivative thereofand/or a nucleic acid encoding one or more peptides as disclosed hereinor a mutant, variant, analog or derivative thereof, e.g., encapsulationin liposomes, microparticles, microcapsules, recombinant cells capableof expressing the compound, and receptor-mediated endocytosis (see,e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432). Methods ofintroduction can be enteral or parenteral and include but are notlimited to intradermal, intramuscular, intraperitoneal, intravenous,subcutaneous, pulmonary, intranasal, intraocular, epidural, and oralroutes. The agents may be administered by any convenient route, forexample by infusion or bolus injection, by absorption through epithelialor mucocutaneous linings (e.g., oral mucosa, rectal and intestinalmucosa, etc.) and may be administered together with other biologicallyactive agents. Administration can be systemic or local.

In a specific embodiment, it may be desirable to administer thepharmaceutical compositions of the invention locally to the area in needof treatment; this may be achieved, for example, and not by way oflimitation, by local infusion during surgery, topical application, e.g.,by injection, by means of a catheter, or by means of an implant, theimplant being of a porous, non-porous, or gelatinous material, includingmembranes, such as sialastic membranes, fibers, or commercial skinsubstitutes.

In another embodiment, the active agent can be delivered in a vesicle,in particular a liposome (see Langer (1990) Science 249:1527-1533). Inyet another embodiment, the active agent can be delivered in acontrolled release system. In one embodiment, a pump may be used (seeLanger (1990) supra). In another embodiment, polymeric materials can beused (see Howard et al. (1989) J. Neurosurg. 71:105).

Thus, a wide variety of gene transfer/gene therapy vectors andconstructs are known in the art. These vectors are readily adapted foruse in the methods of the present invention. By the appropriatemanipulation using recombinant DNA/molecular biology techniques toinsert an operatively linked polypeptide encoding nucleic acid segmentinto the selected expression/delivery vector, many equivalent vectorsfor the practice of the methods described herein can be generated.

Other Embodiments

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions. Such embodiments are also within thescope of the following claims.

The disclosure also contemplates an article of manufacture, which is alabeled container for providing the one or more peptides as disclosedherein or a mutant, variant, analog or derivative thereof. An article ofmanufacture comprises packaging material and a pharmaceutical agent ofthe one or more peptides as disclosed herein or a mutant, variant,analog or derivative thereof, contained within the packaging material.

The pharmaceutical agent in an article of manufacture is any of thecompositions of the present invention suitable for providing the one ormore peptides as disclosed herein or a mutant, variant, analog orderivative thereof and formulated into a pharmaceutically acceptableform as described herein according to the disclosed indications. Thus,the composition can comprise the one or more peptides as disclosedherein or a mutant, variant, analog or derivative thereof or a DNAmolecule which is capable of expressing such a peptide.

The article of manufacture contains an amount of pharmaceutical agentsufficient for use in treating a condition indicated herein, either inunit or multiple dosages. The packaging material comprises a label whichindicates the use of the pharmaceutical agent contained therein.

The label can further include instructions for use and relatedinformation as may be required for marketing. The packaging material caninclude container(s) for storage of the pharmaceutical agent.

As used herein, the term packaging material refers to a material such asglass, plastic, paper, foil, and the like capable of holding withinfixed means a pharmaceutical agent. Thus, for example, the packagingmaterial can be plastic or glass vials, laminated envelopes and the likecontainers used to contain a pharmaceutical composition including thepharmaceutical agent.

In preferred embodiments, the packaging material includes a label thatis a tangible expression describing the contents of the article ofmanufacture and the use of the pharmaceutical agent contained therein.

EXAMPLES Example 1 Testing of Anti-Inflammatory Potential in a MouseSepsis Model

The ability of peptide drugs was tested to for their potential for thereduction of inflammatory cytokines following treatment with purifiedbacterial lipopolysaccharide (LPS)—a major component of bacterial cellwall of Gram-negative bacteria. A model of inflammation was used whereinmice were injected with LPS, which induced a transient immune responseand a surge of serum inflammatory cytokines. The immune response peaksafter 90 minutes and subsides after 24 hours. The levels of key seruminflammatory markers were compared between mice treated and untreatedwith LPS 90 minutes after injection.

We designed 18 different peptides based on the hAAT sequence (Accession#AAB59371), and had these synthesized using conventional FMOC chemistry.To assess in vivo anti-inflammatory and therapeutic potential, thepeptides were tested in a mouse lipopolysaccharide (LPS) challenge modelwith serum TNFα levels as readout. Using dexamethasone as reference,groups of 3 animals were IP injected with 0.2 mg/kg peptide 2 hoursprior to an LPS challenge, and serum TNFα levels determined 30 min afterthe LPS challenge, by ELISA. The best performing peptide reduced serumTNFα levels equivalent to those achieved with 1 mg/kg dexamethasone.Next, shortened versions of this peptide were synthesized, tested in themouse LPS challenge model, and a 17 amino acid peptide, SP16, emerged asa lead development candidate for further characterization. Next, SP16was tested in a lethal endotoxemia model, which models endotoxemiafollowing acute radiation exposure, where Gram negative bacteria leakingfrom the gastro-intestinal system can cause lethal endotoxemia. In thisexperiment, peptide treatment improved survival (FIG. 7). In vitro, SP16lowers LPS-induced NFκB activation in THP1 cells, consistent with the invivo anti-inflammatory effect.

Mice were injected with 0.5, 0.1, 0.02 and 0.004 mg of each peptide.Mice were also treated with dexamethasone as a positive control andvehicle as a negative control. Two hours later each mouse was injectedwith LPS. Samples were taken 90 minutes after LPS injection.

Results are shown in FIGS. 3 and 4. FIG. 3 shows TNF-α levels from bloodin mice injected with each amount of each peptide. FIG. 4 shows only theresults for mice injected with 0.004 mg of each peptide.

The results shows that the peptides described herein are effective indecreasing TNF-α levels and thus are effective in reducing inflammationin mammalian subjects.

Example 2 Alanine Screened Peptides in Sepsis Mouse Model

To evaluate the effect of different amino acids in the SP16 peptide forthe immunomodulating effect, we created an alanine screen. The differentpeptides in the alanine screen are shown in Table B. FIG. 10 shows thatboth the N- and C-terminally alanine-substituted peptides, where the 3most terminal amino acids had been replaced with alanine, lost most oftheir ability to reduce TNF-alpha levels. The peptides wherein thesubstitution occurred within amino acids 4-14 of SP16, appeared tomaintain and rather improve their capacity to reduce TNF-alpha levelscompared to the control Dexamethasone. The LPS mouse model is anestablished model for sepsis in humans and thus we conclude thatpeptides wherein the amino acids 1-3 and 15-17 are present, can beeffectively used in humans to treat or prevent sepsis in conditions,such as burns or acute radiation, which expose humans to a high risk ofdeveloping endotoxemia.

Example 3 Testing of Anti-Inflammatory Potential in a Collagen AntibodyInduced Arthritis (CAIA) Model of Rheumatoid Arthritis

The ability of peptide drugs was tested to for their potential to reduceinflammation and paw edema in a mouse arthritis model: the CAIA model.Balb/c mice were injected with a collagen antibody cocktail on Day 0 andreceived an LPS boost on Day 3. Following the LPS boost, the pawswelling was scored for each paw. For the results shown in FIG. 5,animals were dosed with 0.2 mg/kg of the peptide set forth in SEQ IDNO:1 (also referred to as SP16), daily. In FIG. 6, Balb/c mice wereintravenously injected with a collagen antibody cocktail (MDBioSciences) on Day 0 and intraperitoneally injected with LPS on Day 3.Paw swelling was determined on Days 0, 3, 4, 5, 6 and 7, and the graphshows cumulative scores for all paws of each experimental group of 5animals. Untreated control did not receive the CAIA cocktail or LPS. Themock group received both the antibody cocktail and LPS boost.Dexamethasone was administered daily at 1 mg/kg. The peptide set forthin SEQ ID NO:1 was dissolved in water and administeredintraperitoneally, at 0.6 mg/kg daily, or a one-time dose of 0.6 mg/kgon day 3.

FIG. 9 shows efficacy of SP16 in the preclinical CAIA mouse model of RA.Graph summarizes data from a study in the mouse CAIA RA model. The graphshows cumulative swelling scores for all paws at the peak of disease(Day 7) for groups of 5 animals. Balb/c mice were injected intravenouslywith a collagen antibody cocktail (MD Biosciences) on Day 0 and injectedintraperitoneally with LPS on Day 3. Normal control animals received noinjections and served as disease-free baseline control. We show thatdaily SP16 injection provided protection equivalent to Dexamethasone.

As shown in FIGS. 5, 6, and 9 the peptide: VKFNKPFVFLMIEQNTK (SEQ IDNO:1) is an effective anti-inflammatory and/or immune-modulating agentin the sepsis model.

Example 4 SP16 Improves Glycemic Control in the db/db Model Type IIDiabetes

Encouraged by the sepsis and RA data, we hypothesized whether SP16 wouldprotect db/db mice, a type II diabetes (T2DM) model, from gluco- andlipotoxicity induced β-cell loss and reduce insulin resistance (25, 39).To test this hypothesis, a study was designed and executed in db/dbanimals. We started a study in the db/db model of T2DM because it iseasier to manage (due to timing and synchronized onset of overtdiabetes) and thus more cost effective than a study in NOD mice.

In the db/db model, SP16 treatment resulted in lowered non-fasted bloodglucose and HbA1c levels, increased C-peptide levels and improvedglucose tolerance (FIG. 8). Non-fasted blood glucose and glucosetolerance were improved over the vehicle control group, although theRosiglitazone group showed better values than the SP16 group. Together,these data show that SP16 treatment improved glycemic control in anestablished type II diabetes model.

These data, together with the Sepsis and RA results, show that SP16possesses the anti-inflammatory and immune-modulating properties of theparent protein hAAT, thus providing a much more cost-effective way oftreatment as the size of the newly discovered peptide fragment issignificantly smaller than that of hAAT.

Example 5 SP16 Peptide is a Toll Like Receptor-2 Agonist

Toll-like receptor 2 (TLR-2) plays a role in the immune system and it isa member of the Toll-like receptor (TLR) family which plays afundamental role in pathogen recognition and activation of innateimmunity. TLR-2 gene is expressed most abundantly in peripheral bloodleukocytes, and has been shown to mediate host response to Gram-positivebacteria and yeast via stimulation of NF-kappaB.

Our data from engineered cell lines show that SP16 activates the TLR-2signaling pathway, but not TLR-4 signaling. This is interesting becauseanother immune modulating peptide, DiaPep277, which shares no sequencesimilarity with SP16, has a similar TLR activation profile. Withoutwishing to be bound by a theory, based on these observations, we suggestthat SP16 acts through the TLR2 receptor, and possible the T-cellreceptor, to drive cytokine secretion to a Th2 anti-inflammatorycytokine profile (IL-4 and IL-10). In autoimmune diseases, SP16 ispredicted to induce expansion of regulatory T-cell populations andthereby shift the inflammatory response towards a regulatory response.

Specifically, FIG. 11 shows that SP16 is a TLR2 agonist. Graphsummarizing data from an experiment with an engineered TLR-2 indicatorcell line (HEK-BLUE™ mTLR2, Invivogen). Cells were incubated with theindicated concentrations of peptide for 24 hours. Upon TLR2 activation,the cells secrete alkaline phosphatase which can be assayed. The assaywas done in triplicate and averages with standard deviations areplotted. SP16 exhibited TLR-2 ligand properties, inducing TLR-2signaling in a dose dependent manner. The a scrambled control peptide(SP34) showed no TLR2 induction. *p<0.05, compared with scrambledcontrol (SP34).

FIG. 12 shows structure activity relationship analysis for SP16. Graphsummarizing data from an experiment where an engineered TLR-2 indicatorcell line (HEK-Blue™ mTLR2, Invivogen) was used to test the impact ofsubstituting amino acid residues of the SP16 peptide with alanine(“alanine scan”). Cells were incubated with 20 μg/ml of the indicatedpeptides for 24 hours. Upon TLR2 activation, the cells secrete alkalinephosphatase which can be assayed. The assay was done in triplicate andaverages are plotted. Peptide sequences are shown in the followingfigure. *p<0.05, compared with scrambled control (SP34).

FIG. 13 shows structure activity relationship analysis for SP16. Tableshowing the amino acid sequences of peptides that were tested using aTLR-2 indicator cell line (See data in FIG. 12). The right side of thetable summarizes the peptides' impact on TLR-2 signaling (* indicateslow, ***** indicates high, N/A had no impact on signaling).

The data suggest the first three residues contribute to inducing TLR-2signaling. If residues 1-3 are substituted with alanines (SP37), themutant peptide has no impact on TLR2. However, when substitutedindividually (SP52-SP54), the peptides retain the ability to stimulateTLR-2. Surprisingly, substitution of the phenyl alanine residue atposition 3 with a smaller alanine residue enhances the ability tostimulate TLR-2 signaling compared to SP16.

Example 6 Oral Administration

FIG. 16 shows a graph summarizing data from a study in the mouse CAIARheumatoid Arthritis model. The graph shows the average cumulativeswelling (clinical) scores for all paws at the peak of disease (Day 7)for groups of 5 animals. Balb/c mice were IV injected with a collagenantibody cocktail (MD BioSciences) on Day 0 and IP injected with LPS onDay 3. Normal Control Animals received no injections and served asdisease-free baseline control. SP16 was provided daily byintraperitoneal injection (Dose: 12 ug/animal) or by oral gavage (Dose:25 or 50 ug/animal). Daily SP16 injection provided protection equivalentto daily administration of 1 mg/kg Dexamethasone.

Example 7 In Vitro Data

FIG. 17 depicts a graph with data from an experiment with mouse RAW(macrophage) cells. Cells were incubated with 20 or 40 ug/ml of SP16peptide, as well as 0, 2.5, 5 or 10 ng/ml LPS, for 24 hours. Upon LPSstimulation, the cells secrete the inflammatory cytokine IL-6, which wasmeasured by ELISA. The assay was done in triplicate and averages withstandard deviations are plotted. SP16 lowered LPS-induced IL-6 secretionconsistent with its anti-inflammatory function. (*) indicates p<0.05compared to scrambled peptide control.

Example 8 Treatment of NOD Model of Diabetes

NOD (non-obese diabetic) mice spontaneously develop overt,insulin-dependent type I diabetes. The NOD model is recommended by theFDA for preclinical testing of therapeutic agents for type I diabetes.Disease onset typically occurs at 12-14 weeks of age, withperi-insulitis detectable from about 4 weeks of age and developing intosevere insulitis at 10 weeks of age. The autoimmune destruction ofβ-cells is thought to arise from dysregulation of multiple tolerancepathways, leading to islet infiltration with monocytes, predominantlyCD4⁺ and CD8⁺ T cells, and insulitis, with subsequent hypoinsulinemiaand hyperglycemia. Administration of the SP16 peptide can delay and/orprevent development of type I diabetes in NOD mice similar to hAATtreatment (FIG. 18).

We claim:
 1. A method of improving glycemic control in a subject havinghyperglycemia comprising administering to the subject havinghyperglycemia an oral formulation comprising a peptide selected from thegroup consisting of: (a) a peptide consisting of the amino acid sequenceX1-Z1-F-N-K-P-F-X2-Z2-X3-Z3-Q (SEQ ID NO: 2), wherein X1 is V or L; X2is V, L or M; X3 is M, I or V; Z1 is any amino acid; Z2 is a sequence ofany two amino acids; and Z3 is a sequence any five amino acids, andwherein the peptide is 37 or fewer amino acids; (b) a peptide consistingof the amino acid sequence VKFNKPFVFLMIEQNTK (SEQ ID NO: 1); (c) apeptide consisting essentially of the amino acid sequenceX1-Z1-F-N-X2-P-F-X3-Z2-X4-Z3-X5 (SEQ ID NO: 3), wherein X1 is V or L; X2is K or R; X3 is V, L or M; X4 is M, I or V; X5 is K or Q; Z1 is anyamino acid; Z2 is a sequence of any two amino acids; and Z3 is asequence any five amino acids; (d) a peptide consisting essentially ofthe amino acid sequence RFNRPFLR (SEQ ID NO: 4). (e) a peptideconsisting essentially of the amino acid sequence of RRRFNRPFLRRR (SEQID NO: 8). (f) a peptide consisting essentially of the amino acidsequence of VKFNKPFVFLMIEQNTK (SEQ ID NO: 1); and (g) a peptideconsisting essentially of the amino acid sequence of FNRPFL (SEQ ID NO:10).
 2. The method of claim 1, wherein the peptide further comprises atleast one second peptide or protein.
 3. The method of claim 2, whereinthe at least one second protein or peptide is attached to the peptide asa fusion peptide.
 4. The method of claim 2, wherein the at least onesecond peptide or protein is an epitope tag or a half-life extender orboth.
 5. The method of claim 1, wherein the peptide comprises one ormore D-amino acids.
 6. The method of claim 1, wherein the subject havinghyperglycemia is affected with type II diabetes or type I diabetes. 7.The method of claim 6, wherein the inflammatory condition is type Idiabetes.
 8. The method of claim 1, wherein the peptide causes a 75%decrease in serum TNF-α levels when administered in an effective amountto a human subject.
 9. The method of claim 1, wherein the peptideconsists of 35 amino acid residues or fewer.
 10. The method of claim 1,wherein the peptide consists of 22 amino acid residues or fewer.
 11. Themethod of claim 1, wherein the peptide consists of 21 amino acidresidues or fewer.
 12. The method of claim 1, wherein the compositionfurther comprises a pharmaceutically acceptable carrier.
 13. A method ofdecreasing serum TNF-α levels in a human subject comprisingadministering to the subject an oral formulation comprising a peptideselected from the group consisting of: (a) a peptide consisting theamino acid sequence X1-Z1-F-N-K-P-F-X2-Z2-X3-Z3-Q (SEQ ID NO: 2),wherein X1 is V or L; X2 is V, L or M; X3 is M, I or V; Z1 is any aminoacid; Z2 is a sequence of any two amino acids; and Z3 is a sequence anyfive amino acids, and wherein the peptide comprises 37 or fewer aminoacids; (b) a peptide consisting the amino acid sequenceVKFNKPFVFLMIEQNTK (SEQ ID NO: 1); (c) a peptide consisting essentiallyof the amino acid sequence X1-Z1-F-N-X2-P-F-X3-Z2-X4-Z3-X5 (SEQ ID NO:3), wherein X1 is V or L; X2 is K or R; X3 is V, L or M; X4 is M, I orV; X5 is K or Q; Z1 is any amino acid; Z2 is a sequence of any two aminoacids; and Z3 is a sequence any five amino acids; (d) a peptideconsisting essentially of the amino acid sequence RFNRPFLR (SEQ ID NO:4). (e) a peptide consisting essentially of the amino acid sequence ofRRRFNRPFLRRR (SEQ ID NO: 8). (f) a peptide consisting essentially of theamino acid sequence of VKFNKPFVFLMIEQNTK (SEQ ID NO: 1); and (g) apeptide consisting essentially of the amino acid sequence of FNRPFL (SEQID NO: 10).
 14. The method of claim 13, wherein the subject isadministered the composition in an amount that results in at least 50%reduction of the TNF-α levels compared to the TNF-α levels in thesubject prior to administering the peptide.
 15. The method of claim 13,wherein reduction of the TNF-α levels is at least 75%.